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This is to certify that the

dissertation entitled

PHYTOCEUTICAL AND OTHER BIOACTIVE NATURAL PRODUCTS

FROM APIUM GRAVEOLENS L. SEEDS

 

presented by

Raf ikali A. Momin

has been accepted towards fulfillment
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Ph.D. degree in Horticulture

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PHYTOCEUTICAL AND OTHER BIOACTIVE NATURAL PRODUCTS
FROM APIUM GRA VEOLENS L. SEEDS
By

Rafikali A. Momin

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Horticulture

2000

ABSTRACT
PHYTOCEUTICAL AND OTHER BIOACTIVE NATURAL PRODUCTS FROM
APIUM GRA VEOLENS L. SEEDS
By

Rafikali A. Momin

Earlier research on Apium graveolens seeds was focused on its phytochemistry and its
application in flavor and fragrances industries. Though, celery seeds are well known for
its medicinal properties, very little work has been done on the isolation and identification
of bioactive compounds. A phytochemical investigation on various solvent extracts of A.
graveolens seeds resulted in the isolation and characterization of mosquitocidal,
nematicidal, antimicrobial, topoisomerase and cyclooxygenase inhibitory and antioxidant
compounds. A. graveolens seeds were sequentially extracted with hexane and methanol.
Bioassay-directed isolation and purification of the hexane extract of A. graveolens seeds
afforded three compounds, B-selinene (1), 3-n-butyl-4,5-dihydrophthalide (2) and S-allyl-
2-methoxyphenol (3). The triglyceride, l,3-di[(cis)—9-octadecenoyl]-2-[(cis,cis)-9,12-
octadecadienoyl]glycerol (4) and compound 3 were isolated for the first time from A.
graveolens seeds. Hexane and CHCI3 soluble fractions of methanolic extract of A.
graveolens seeds yielded bioactive compounds, sedanolide (5), senkyunolide-N (6),
senkyunolide-J (7) and a novel compound, 3-hydroxymethyl-6-methoxy-2,3-dihydro-1H-
indol-2-ol (8). Compounds 6 and 7 were isolated for the first time from A. graveolens.
Antioxidant and cyclooxygenase inhibitory assay directed purification of CHCI3

insoluble fraction of methanolic extract of A. graveolens seeds yielded L-tryptophan (9)

and 7-[3-(3,4-dihydroxy-4-hydroxymethyl-tetrahydro-furan-2-yloxy)-4,5-dihydroxy-6-
hydroxymethyl-tetrahydro-pyran-2-yloxy]-5-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-
chromen-4-one (10). The structures of these compounds were established by using
various spectroscopic experiments such as 1D- and 2D- NMR, FTIR, and MS.

Compounds 1-3 demonstrated 100 % mortality on fourth-instar Aedes aegyptii
larvae at 50, 25 and 200 ug mL", respectively, in 24 h. The LDwo in 24 h for compound
2 was observed at 12.5 and 50 ug mL'l, when tested on nematodes, Panagrellus redivivus
and Caenorhabditis elegans, respectively. Also, it inhibited topoisomerase-I and —II
enzyme activities as well as the growth of Candida albicans and Candida kruseii at 100
ug mL". The triglyceride (4) was not biologically active in our assays.

The 100 % mortality of the nematode P. redivivus was caused by compounds 5-7
at 25, 100 and 100 ug mL'l, respectively. Compound 5 showed 100 % mortality at 50 ug
mI.’I on nematode, C. elegans, and fourth-instar mosquito larvae, A. aegyptii. It also
inhibited the growth of C. albicans and C. parapsilasis at 100 ug mL". Topoisomerase-I
and —II inhibitory activities of compounds 5-7 were recorded at 100, 200 and 200 ug mL'
1 concentrations, respectively. This is the first report of the mosquitocidal, nematicidal,
topoisomerase enzyme inhibitory and antimicrobial activities of compounds 5-7.
Compounds 5-10 were also tested for their cyclooxygenase inhibitory and antioxidant
activities. At pH-7 most of the test compounds demonstrated very good cyclooxygenase
inhibitory activities as demonstrated by the inhibition of prostaglandin H endoperoxide
synthase-I (COX-1) and of prostaglandin H endoperoxide synthase-ll (COX-II) at 250 ug
mL'l concentration. Compound 8 is here identified as a novel compound that showed the

highest COX-1 and —II inhibitory activities. Antioxidant activities of compounds 9 and 10

were comparable to commercially available antioxidants when assayed at 125 and 250 ug
ml.’l concentrations, respectively.

Humans on a daily basis consume many plants. Natural products isolated from the
edible plants with anticarcinogenic and antiinflammatory activities should be readily
acceptable as a dietary supplement if the toxicity and side effects are minimal. Both
antiinflammatory and antioxidant activities of compounds from A. graveolens seeds
suggest that consumption of celery seeds may alleviate pain related to gout and arthritis.
Also, it has potential in the control of nematodes and other agricultural pests as evidenced
by the good nematicidal and mosquitocidal activities of phthalides isolated from celery
seeds. Considering the potential phytoceutical and agriculture pest management
properties of A. graveolens seeds, it may result into novel products which could generate

additional income to celery growers.

TO MY DARGNTS

ACKNOWLEDGMENTS

I would like to express my sincere gratitude and thanks to my major advisor Prof.
Muraleedharan G. Nair for his valuable guidance, constant encouragement, constructive
criticism and the final shaping of this dissertation. The timely completion of this
dissertation would not have been possible without his support, mentoring and patience.

I am grateful to the members of my advisory committee, Drs. Douglas A. Gage,
Randolph M. Beaudry, Gale M. Strasburg and Robert E. Schutzki for their assistance,
valuable suggestions and encouragement through out my study. I also gratefully
acknowledge Drs. Long Lee and Kermit Johnson for their assistance in conducting
various NMR experiments.

I wish to express special thanks to my fellow members and friends, Dr. Seeram,
Dr. Ramsewak, Dr. Balasubramanian, Dr. Wang, Dr. Kelm, Dr. Henry, Jennifer Miles,
Andrew Erickson, Laura Clifford, Di Wu, Robert Cichewicz and Marvin Weil for their
moral support and assistance.

I gratefully acknowledge the financial assistance provided by THE AGA KHAN
FOUNDATION, Geneva, Switzerland during the course of this study.

I owe special thanks to my parents, uncle, aunt, brothers and sister for their love,
kindness, inspiration, encouragement, moral support and blessings. Finally, I must
express deep love and appreciation to my dear wife Almas for her tireless support during
long nights of work away from her, and for her understanding, sacrifice, confidence,

encouragement, enthusiasm and moral support.

vi

TABLE OF CONTENTS

LIST OF FIGURES ...................................................................................................... ix
LIST OF ABBREVIATIONS ....................................................................................... xi
INTRODUCTION ......................................................................................................... l
CHAPTER-1 .................................................................................................................. 6
LITERATURE REVIEW

Pharmacological Studies ................................................................................................ 7
Phytochemical Studies ................................................................................................. 15
Table 1.1 The chemical composition of celery seed oil (Cu et al., 1990) ................... 31
CHAPTER-2 ............................................................................................................... 34

Bioactive compounds and l,3-di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9,l2-
octadecadienoyl]glycerol from Apium graveolens L. seeds

Abstract ....................................................................................................................... 34
Introduction .................................................................................................................. 35
Materials and Methods ................................................................................................. 36
General Experimental Procedures ................................................................................ 36
Plant Material ............................................................................................................... 37
Extraction and Isolation ............................................................................................... 37
Saponification and Methylation of compound 4 and standards ................................... 39
Mosquitocidal Assay .................................................................................................... 40
Nematicidal Assay ...................................................................................................... 4O
Antimicrobial and Topoisomerase-I and —II Inhibitory Assays .................................. 41
Results and Discussion ................................................................................................ 42
CHAPTER-3 ................................................................................................................ 48

Mosquitocidal, nematicidal and antimicrobial compounds from Apium graveolens
L. seeds

Abstract ........................................................................................................................ 48
Introduction .................................................................................................................. 49
Materials and Methods ................................................................................................. 50
General Experimental Procedures ............................................................................... 50
Plant Material ............................................................................................................... 50
Extraction and Isolation ............................................................................................... 51
Mosquitocidal Assay .................................................................................................... 53
Nematicidal Assay ....................................................................................................... 54
Antimicrobial Assay .................................................................................................... 54
Results and Discussion ................................................................................................ 55

vii

CHAPTER-4 ................................................................................................................. 61
Antioxidant, cyclooxygenase and topoisomerase inhibitory compounds from
Apium graveolens Linn. seeds

Abstract ......................................................... 61
Introduction ................................................................................................................. 62
Materials and Methods ................................................................................................ 64
General Experimental Procedures ............................................................................... 64
Plant Material .............................................................................................................. 64
Extraction and Isolation ............................................................................................... 65
Cyclooxygenase Inhibitory Assay .............................................................................. 68
Antioxidant Assay ........................................................................................................ 69
Topoisomerase-I and —II Inhibitory Assay ................................................................. 70
Results and Discussion ................................................................................................ 71
Scheme-l ...................................................................................................................... 73
CHAPTER-5 ................................................................................................................. 85

Summary and Conclusion

LITERATURE CITED ............................................................................................... 90

viii

LIST OF FIGURES

Figure 2.1. Percent mortality of 4‘h instar A. aegyptii larvae for compounds 1-3 at 24 h.
Statistical analysis was done using ANOVA (p 5 0.01) and the means were compared by

calculating least significant difference (LSD) ............................................................. 46

Figure 2.2. Percent mortality of C. elegans and P. redivivus nematodes for compound 2 at
24 h. Statistical analysis was done using ANOVA (p _<_ 0.01) and the means were
compared by calculating least significant difference (LSD) ........................................ 47

Figure 3.1. CD spectra of compounds 6 and 7 in MeOH ............................................. 57

Figure 3.2. Percent nematicidal activity against P. redivivus for compounds 5-7 at 24 h.
Statistical analysis was done using ANOVA (p 5 0.01) and the means were compared by

calculating least significant difference (LSD) ............................................................. 59

Figure 3.3. Percent nematicidal activity against C. elegans for compound 5 at 24 h.
Statistical analysis was done using ANOVA (p 5 0.01) and the means were compared by
calculating least significant difference (LSD) ............................................................. 60

Figure 4.1. HMBC connectivities in compound 8 ....................................................... 72
Figure 4.2. In vitro dose response of COX-1 and COX-ll inhibitory activities of
compound 5. Both ibuprofen and naproxen were tested at 2.06 and 2.52 ug mL'l,
respectively. .................................................................................................................. 77
Figure 4.3. In vitro dose response of COX-1 and COX-II inhibitory activities of

compound 6. Both ibuprofen and naproxen were tested at 2.06 and 2.52 ug mL'l,
respectively. .................................................................................................................. 78

ix

Figure 4.4. In vitro dose response of COX-1 and COX-II inhibitory activities of
compound 7. Both ibuprofen and naproxen were tested at 2.06 and 2.52 ug mL",
respectively. .................................................................................................................. 78

Figure 4.5. In vitro dose response of COX-1 and COX-II inhibitory activities of
compound 8. Both ibuprofen and naproxen were tested at 2.06 and 2.52 ug mL",
respectively. .................................................................................................................. 79

Figure 4.6. In vitro dose response of COX-1 and COX-II inhibitory activities of
compound 9. Both ibuprofen and naproxen were tested at 2.06 and 2.52 ug mL’l,
respectively. .................................................................................................................. 79

Figure 4.7. In vitro dose response of COX-1 and COX-II inhibitory activities of
compound 10. Both ibuprofen and naproxen were tested at 2.06 and 2.52 ug mL",
respectively. .................................................................................................................. 80

Figure 4.8. In vitro dose response of COX-1 and COX-II inhibitory activities of
compound 11. Both ibuprofen and naproxen were tested at 2.06 and 2.52 ug mL",
respectively. .................................................................................................................. 80

Figure 4.9. Antioxidant activities of some commercial antioxidants. Data represent the

mean of duplicate experiments ................................................................................... 82

Figure 4.10. Antioxidant activity at 21 min for some commercial antioxidants. Data

presented are the mean of duplicate experiments ......................................................... 82

Figure 4.11. Antioxidant activities of compounds 5-11. Data represent the mean of

duplicate experiments ................................................................................................... 83

Figure 4.12. Antioxidant activity at 21 min for compounds 5-11. Data presented are the

mean of duplicate experiments ..................................................................................... 83

BHA
BHT
CD
CR}
CHCh
cox
l3CNMR
DMSO

dd
DPA—PA
DEPT
IflMS
E120
BOH
EtOAc
FABMS
FUR
GC
'HNMR
HMBC
HMQC
HPLC
IR

LC

MeOH
MIC

MOPS
MPLC

LIST OF ABBREVIATIONS

Butylated hydroxyanisole

Butylated hydroxytoluene

Circular Dichroism

Colony forming unit

Chloroform

Cyclooxygenase

Carbon nuclear magnetic resonance
Dimethyl sulfoxide

Doublet

Doublet of doublet

3-(p-(6-phenyl)-1 ,3,5-hexatrienyl)phenylpropionic acid
Distortionless enhancement polarization transfer
Electron impact ionization mass spectroscopy
Diethyl ether

Ethanol

Ethyl acetate

Fast atom bombardment mass spectroscopy
Fourier transfer infrared spectroscopy

Gas chromatography

Proton nuclear magnetic resonance
Heteronuclear multiple bond coherence
Heteronuclear correlation through multiple quantum coherence
High pressure liquid chromatography
Infrared

Coupling constant

Lethal concentration

Multiplet

Methanol

Minimum inhibitory concentration
3-[N-morpholino] propanesulfonic acid
Medium pressure liquid chromatography
Mass spectroscopy

Mass-to-charge ratio

Nuclear magnetic resonance

Prostaglandin

Prostaglandin endoperoxide H synthase-l
Prostaglandin endoperoxide H synthase-2
Parts per million

Preparative thin layer chromatography
Reference value

Retention time

Singlet

xi

TBA
TBHQ
THF
TLC
UV
VLC
YMG
YPDA

[91

2—thiobarbituric acid
tert-butylhydroquinone
Tetrahydrofuran

Thin layer chromatography
Ultraviolet

Vacuum liquid chromatography
Yeast maltose glucose

Yeast potato dextrose agar
Chemical Shifts

Molar ellipticity (in Circular dichroism)
wavelength

xii

INTRODUCTION

Apium graveolens L., celery, is a hapaxanthic herb, grown as a biennial and under certain
conditions, as an annual. It is a native of Eurasia and grown as a wild plant. It prefers
soils containing sodium chloride and, therefore, was grown in mainly coastal regions.
Today, celery is widely cultivated in the temperate zones as an important garden crop and
the bleached leaf stalks being relished as a popular vegetable. Countries leading in
production of celery are France, India, and the USA. Celery, known in Hindi as Ajmud,
grows widely in the foot of northwest Himalayas and outlying hills in the Punjab and
Uttar pradesh. In India, celery with its various names like Ajamoda, Bastamoda,
Kharaashwa, Mayoori, Deepa, Karavee, Lochamostaka is well known for the treatment of
a variety of ailments. In the USA, celery was first produced commercially in 1856 in
Kalamazoo, Michigan. Celery fruit, commonly known as “celery seed”, was largely
imported from Europe. Since World War-II, celery has been produced as a domestic crop,
especially in Michigan and Wisconsin, and distributed throughout the USA. Celery can
be classed both as an ingredient for seasoning and as a vegetable. For seasoning
purposes, the seeds and the plant, as well as the essential oil distilled from them, are used
(Salzer, 1975). The stalks of the celery plant are eaten raw in salads or cooked as in
soups or in stir-fried foods. Celery is also used for its unique texture and appetizing

flavor.

The celery seed is ovate, has a dark brown cremocarp and possesses a

characteristic aroma with somewhat pungent taste. It is employed as a condiment in the

flavoring of food products. Celery seed oil, celery seed, and celery seed extracts are all
extensively used as flavoring ingredients in all major food products, including alcoholic
and nonalcoholic beverages, frozen dairy desserts, candy, baked goods, gelatins and
puddings, meat and meat products, condiments and relishes, soups, gravies, snack foods,
and others. Celery seed oil is reportedly used as diuretic, in dropsy and bladder ailments,
antispasmodic, and in rheumatoid arthritis (Styavati and Raina, 1976; Chopra, et al.,
1958). In European tradition celery seeds were reportedly used as carminative, stomach
ache, diuretic, laxative, for glandular stimulation, gout, kidney stones, rheumatic
complaints, loss of appetite and exhaustion. Leaves and petioles of celery were also used
for skin problems in addition to the above mentioned uses. In India, celery seeds are used
for treating bronchitis, asthma, liver and spleen diseases (Chopra, et al., 1958, Satyavati
and Raina, 1976). Polyherbal formulations reputed to have hepatoprotective activity are
available in the Indian market, which comprise about more than one hundred Indian
medicinal plants including celery seed. Celery is an ingredient of several herbal

formulations with reputed liver—protecting activities (Handa et al., 1986).

Earlier research on A. graveolens seeds was focused primarily on their
phytochemistry and its application in both the flavor and fragrances industries. Celery is
also a well known folk medicine with a variety of medicinal properties. For these reasons,
celery seed oil has been studied for its compositions (Vemin and Parkanyi, 1994). Most
of these studies on A. graveolens seed extracts were focused on the bioactivity of crude
extracts. Very little work has been done on the isolation and identification of bioactive

compounds from A. graveolens seeds. Compounds present in celery seeds, which might

contribute to the anticarcinogenic, antiinflammatory, antioxidant and other bioactivities

need to be characterized.

Studies on various biological activities of crude extracts, essential oils and pure
compounds isolated from A. graveolens are discussed in Chapter-1. A brief review of
the phytochemical constituents of A. graveolens seeds is also discussed in Chapter-1.
The phthalides, naturally occurring in edible umbeliferous plants may be useful
chemopreventive against carcinogenesis and tumorigenesis (Zheng et al., 1993).
However, additional research must be performed to check the anticarcinogenic effect of
the phthalides occurring in celery seeds. Al-Hindawi and co-workers (1989) observed
antiinflammatory activity in the ethanol extract of celery plant, but they could not
establish any link between known constituents with the observed antiinflammatory
activity. The antiinflammatory activities of compounds present in celery seeds need to be
investigated. Also, many other constituents present in celery seeds, which might
contribute to the anticarcinogenic, antiinflammatory, and antioxidant activities of celery
seeds need to be characterized. Therefore, it is my hypothesis that given the body of pre-
existing research on celery seeds and preliminary studies carried out in the Bioactive
Natural Products Laboratory (BNPL), A. graveolens seeds have the potential to yield
novel compounds with anticarcinogenic, antiinflammatory, antioxidant, antimicrobial,
mosquitocidal and nematicidal activities. This could yield phytoceuticals or
agrochemicals and result in additional income to celery growers in Michigan and USA.
In order to test this hypothesis, I have conducted a bioassay-directed isolation and
characterization of compounds in celery seed extracts by using cyclooxygenase enzymes

and Fe2+ -induced lipid peroxidation bioassays. These compounds were also evaluated for

antimicrobial, mosquitocidal and nematicidal activities according to published bioassay
procedures (Nair, et al., 1989; Roth, et al., 1998). Therefore, the objectives of my
research were to conduct bioassay-directed isolation and identification of compounds in
celery seeds using chromatographic and spectral methods, and determine the
anticarcinogenic, antiinflammatory, antioxidant, antimicrobial, nematicidal and
mosquitocidal efficacies of purified compounds. In addition, it is expected that the
present work should add to the existing knowledge on the bioactive constituents in A.

graveolens.

A. graveolens seeds were provided by Asgrow Seed Company, Kalamazoo,
Michigan and stored at —20 0C until extraction. Initially the crude extracts of A.
graveolens have been examined for insecticidal, nematicidal, antimicrobial and
pharmacological studies. Based on the initial bioassay results, bioassay-directed isolation
and purification of compounds from A. graveolens seeds were performed. I examined
crude extracts and pure compounds of celery seeds for their biological activities. Insect
bioassay utilizing mosquito larvae (Aedes aegyptii Linn.) and nematodes, Caenorhabditis
elegans and Panagrellus redivivus, were cam'ed out to determine such activities for
celery compounds. The test organisms Candida kruseii (MSU strain), C. albicans (MSU
strain) and C. parapsilasis (MSU strain) were used for antimicrobial bioassays and
Saccharomyces cerevisiae mutant cell cultures, .IN394, JN394t-1, and JN394t-2-5 were
used for the topoisomerase enzyme inhibitory assay on crude extracts and purified
compounds. Antiinflammatory and antioxidant assays were conducted on crude extracts
and pure compounds using cyclooxygenase enzymes (COX-1 and COX-II) inhibitory and

F 62+ -induced lipid peroxidation assays, respectively.

This dissertation comprises of five chapters and each chapter except the review of
literature is organized as a scientific journal article, which contains an abstract,

introduction, material and methods, and results and discussion section.

Chapter 1

LITERATURE REVIEW

The breadth of literature on the bioactivities of crude extracts and pure compounds from
A. graveolens seeds is reviewed in this chapter. Interesting biological activities related to
human health for compounds from A. graveolens seeds were anti-carcinogenic,
chemopreventative, antiplatelet, anti-inflammatory, diuretic, hepatoprotective, sedative,
vasodilatory, antirheumatic, carminative, antispasmodic, effect on blood pressure,
tranquillizing, anticonvulsant, antioxidant, and hypotensive activities. Other potential
applications in agriculture for A. graveolens seeds reported were antifungal and
antibacterial activities. Very few papers have been published about bioactivities of pure
compounds from A. graveolens, which included anti-carcinogenic, chemopreventative,
inhibition of platelet aggregation, vasodilatory, and antimicrobial activities. This chapter

also covers the phytochemical studies on A. graveolens.

Pharmacological Studies

A wide spectrum of secondary products present in dietary supplements has been
reported to be associated with protective effects against chemically induced toxicity and
carcinogenesis (Hayatsu et al., 1988; Hocman, 1989; Morse and Stonre, 1993). Celery
seeds are used in India to treat bronchitis, asthma, liver and spleen diseases (Styavati and
Raina, 1976). Polyherbal formulations reputed to have hepatoprotective activity and
those are available on the Indian market comprise about one hundred Indian medicinal
plants. A. graveolens is an ingredient of some of these polyherbal formulations having

reputed life-protecting activities (Handa et al., 1986).

Naturally occurring anticarcinogenic components from edible plants have been an
important source for obtaining potential cancer chemopreventive agents. A. graveolens
seed extracts and pure compounds have been shown to possess anticarcinogenic activities
(Zheng et al., 1993; Lam and Zheng, 1992; Banerjee et al., 1994). Zheng et a1. (1993)
studied the chemoprevention of Benzo [a]pyrene-induced fore-stomach cancer in mice by
natural phthalides from A. graveolens seed oil. Zheng and co-workers (1993) had
isolated five natural products, d-limonene, p-mentha-2,8-dien-l-ol, p-mentha-8(9)-en-
1,2-diol, 3-n-butyl phthalide and sedanolide from celery seed oil. However, only p-
mentha—2,8-dien-l-Ol, 3-n-butyl phthalide and sedanolide exhibited appreciable activities
by inducing the detoxifying enzyme glutathione S-transferase (GST) in the target tissues
of female A/J mice. A total of 3 doses of 20 mg/dose for every two days of 3-n-butyl
phthalide, sedanolide, and p-mentha—2,8-dien-1-ol increased the GST activity by 4.5-5.9,

3.2-5.2, and 3.7 times over the controls in mouse liver and small intestinal mucosa,

Fit“

respectively. These five compounds were also tested for their ability to inhibit
benzo[a]pyrene- (BP) induced tumorigenesis in mice. 3-n-butyl phthalide and
sedanolide reduced the tumor incidence from 68 to 30 and 11%, respectively (Zheng et
al., 1993). These results indicated about 55% of inhibition of tumorigenesis after the
treatment of mice with the phthalides. The tumor multiplicity was also significantly
reduced as a result of treatment with these phthalides from celery seeds. Sedanolide and
3-n-butyl phthalide reduced more than 83 and 67% of tumor multiplicity in mouse,

respectively.

Lam and Zheng (1992) studied the effect of essential oils on glutathione S-
transferase (GST) activity in mice including the essential oil from A. graveolens seeds.
Celery seed oil was one of the most active essential oils studied in this experiment. In this
study, the induction of GST enzyme in the cytosols of liver, forestomach, and small
intestinal mucosa was examined. The GST enzymes were catalyzed by the conjugation of
glutathione with electrophilic species of celery seed oil components to form less toxic,
water soluble substances that are readily excreted (Chasseaud, 1979). The enhancement
of GST activity suggested an increase in the host’s ability to detoxify xenobiotics,
including carcinogens. Thus, substances that can elicit increased activity of GST and
other detoxifying enzymes are considered to be a potential anticarcinogens and
chemoprotecting agents (Lam and Zheng, 1992). Essential oil from celery seed given
orally to mice resulted in a 3.29, 3.28 and 1.49 fold increase in GST activities compared
to the control in small intestinal mucosa, liver, and forestomach, respectively. Since an

increase of GST activity suggests anticarcinogenic potential, the celery seed oil is

expected to contain components that may be useful as chemopreventive agents. Banerjee
et a1. (1982) investigated the influence of essential oils derived from several edible plants
such as celery seed (Apium graveolens), cumin seed (Cuminum cyminum), cardamom
(Elettaria cardamonum), coriander (Coriandrum sativum), ginger (Zingiber oflicinale),
nutmeg (Myristica fragrans) and zanthoxylum species on the activities of hepatic
carcinogen-metabolizing enzymes like cytochrome P450, aryl hydrocarbon hydroxylase
and GST and acid-soluble sulfliydryl levels in Swiss albino mice. The celery seed oil
caused approximately 2-fold increase in cytosolic GST enzyme activity (Banerjee et al.,
1982). Also, GST enzyme activity was elevated in livers of Swiss albino mice by celery
seed oil and concluded the enhancement of GST enzyme activity as an indication of its
potential role as “blocking agents” in chemopreventive strategies. There were no
significant increases in acid-soluble sulfliydryl levels and cytochrome P450 enzyme
activity in the liver of mice treated with celery seed essential oil. Though the elevated
sulfhydryl concentration minimizes the adverse effect of toxic xenobiotic compounds,
reduction of the tumorigenic response (Wattenberg, 1985; Shenoy and Choughuley,
1992) and induction of microsomal cytochrome P450 and aryl hydrocarbon hydroxylase
are suggestive of their participation in activation of lipophilic xenobiotics, including

procarcinogens.

The essential oil from A. graveolens has a sedative effect upon the central
nervous system (Guenther, 1961). The tranquilizing and anticonvulsant activities of the
alkaloid fraction obtained from the seeds of A. graveolens were studied by Kulshrestha
et al. (1970). Potentiation of pentobarbital narcosis, amphetamine group toxicity,

conditioned avoidance response (CR), and spontaneous motor activity (SMA) tests were

performed to study the tranquilizing activity of a 2% aqueous sulphuric acid extract of A.
graveolens in albino mice and rat models. In these tests, the ED50 values (in mg per 100
g) were found to be 33.6, 29.8, 24.3, and 68.2 in potentiating pentobarbital narcosis,
conditioned avoidance response, spontaneous motor activity, and amphetamine group
toxicity tests, respectively. Anticonvulsant activity against strychnine convulsions was
tested for celery seed extracts by maximal electroshock seizure, metrazol seizure
threshold and strychnine convulsions tests (Swinyard et al., 1952). The A. graveolens
extract provided protection against only electroshock (EDSO- 34.6 mg/ 100 g). In
conclusion, the low toxicity and high protective index in animal experiments suggested
the therapeutic use of A. graveolens extract in depressant effect on the central nervous
system (Kulshrestha et al., 1970). Some anti-depression effects, the tranquillizing and
anticonvulsant activities of A. graveolens essential oil from seeds, were examined in mice
by Kohli et a1. (1967). The tranquillizing and anticonvulsant action of an essential oil
fraction from celery seed have been demonstrated by using standard methods. The
maximum central activity was found out to be in a fraction collected between boiling
points of 180 and 265 0C. These results supported the sedative effect of celery seeds
upon the central nervous system (0301 and F arrar, 1955). 3-n-butylphthalide, one of the
celery seed constituents was shown to have anticonvulsant effects in experimental
chronic epilepsy induced by coriaria lactones in rats (Yu et al., 1988a). The
anticonvulsant effects of 3-n-butylphthalide were weaker than those of diazepam, but its
ability in counteracting the learning and memory impairment was greater than that of
diazepam, causing no damage to brain cells (Yu, et al., 1988a; Yu et al., 1988b). Same
authors had reported earlier that 3-n-butylphthalide has low acute and chronic toxicities

and no teratogenic activity in experimental animals (Yu, et al., 1988a).

10

 

Celery seeds are claimed to have diuretic effects (Houghton and Manby, 1985).
The phthalides from celery seeds were shown to have diuretic, antispasmodic and anti
convulsion activities (Chevallier, 1998; Boulos, 1983). The volatile oil from A.
graveolens seeds showed similar activities and was reported to imitate the epithetical
tissue of the kidneys, which may cause diuresis. Houghton and Manby (1985) also noted
that the flavonoids present in celery seeds may also play some part in diuresis since it is
known that they inhibit some of the enzymes in the kidney involved in water
reabsorption. Chevallier (1998) has also mentioned that celery seeds have a mildly
diuretic and significantly antiseptic action. Flavonoids are an effective treatment for
crystitis, helping to disinfect the bladder and urinary tubules. Contrary to these reports,
Mahran et al. (1991) found that the volatile oil of A. graveolens seeds in doses of 4 and 8
uL kg'1 body weight did not affect urine flow or blood pressure in dogs. Higher doses of
volatile oil produced hypotensive response. The ethanolic extract of A. graveolens could
not be tested pharmacologically for diuretic property because it produced a gelatinous
material upon dissolving in saline (Mahran et al., 1991). Also, there was no significant
change in urine flow, Na+, K+ or Cl' excretion for more than 2 h in celery seed volatile
oil-treated dogs. Boulos (1983) in his book, “Medicinal Plants of North Africa”
mentioned the diuretic property and effect of celery fruit on kidneys and bladder. The
tincture prepared from matured seeds was used to treat urine retention and other renal
disorders (Boulos, 1983).

The ethanol extract of the aerial parts of A. graveolens was assessed for its anti-
inflammatory activity on intact rats by measuring the suppression of carrageenan-induced
paw edema produced by 1/10 of the intraperitoneal LD50 doses (Al-Hindawi et al., 1989).

Acetylsalicylic acid at doses of 100 and 200 mg kg’1 body weight was used as a positive

11

control and the results were in agreement with the known activity of acetylsalicylic acid.
The inhibition of carrageenan-induced edema in the rat paw after the intraperitoneal
injection of 200 and 358.7 mg g'1 of acetylsalicylic acid and A. graveolens extract were
66.8 and 53.0%, respectively (Al-Hindawi et al., 1989). However, any link between
known constituents in A. graveolens with the observed anti-inflammatory activity was not
established. Lewis et al. (1985) also studied the anti-inflammatory activity of aqueous
extracts of celery stem against two animal models, carrageenan and mouse ear tests.
Indomethacin was used as a positive control in both models. Ethanol and aqueous
extracts of celery stem were used for this study. Both celery powder (400 mg kg“) and
indomethacin (5 mg kg") were anti-inflammatory in the carrageenan model (Lewis et al.,
1985). At a lower dose (100 mg kg“), celery powder showed 30% inhibition of
carrageenan-induced inflammation. Celery powder also showed anti-inflammatory
activity in the mouse ear test. The ethanol extract was not anti-inflammatory but the
celery powder before ethanol extraction inhibited carrageenan-induced inflammation by
71% when measured 5h after the administration of carrageenan. Celery powder residues
after ethanol extraction inhibited carrageenan-induced edema by 62%. Two constituents
of celery stem, manitol and phytosterols (indosterol), were also studied for their anti-
inflammatory activity in these two models (Al-Hindawi et al., 1989). An oral
administration of manitol at a dose of 20 mg kg'I did not show antiinflammatory activity.
The phytosterols from celery showed antiinflammatory activity when administered orally
to rats in an aqueous suspension (Al—Hindawi et al., 1989). Therefore, the observed anti-
inflammatory activity in animal models confirmed the basis for the anecdotal claims of
celery as a medicinal plant for controlling rheumatic disease (Chopra et al., 1958,

Satyavati and Raina, 1976).

12

Platelets play an important role in the hemostatic process and their abnormal
activation will cause arterial thrombosis. Teng et a1. (1988) found the inhibitory effect of
apigenin, a constituent of A. graveolens on platelet aggregation. The effect of apigenin on
collagen-induced platelet aggregation and ATP release, aggregation induced by ADP,
arachidonic acid, platelet activating factor (PAF), thrombin and ionOphore A23187, the
thromboxane formation in platelets, and aggregation of whole blood was studied by Teng
et al. (1988). In this study, apigenin inhibited the aggregation of washed rabbit platelets
induced by collagen, ADP, arachidonic acid and PAF, but not that induced by thrombin
or ionOphore A23187. It also inhibited the release of ATP from platelets induced by all
aggregants used as well as the formation of thromboxane B2 in platelets challenged by
collagen and arachidonic acid. The IC50 on collagen-induced platelet aggregation was
about 50 ug mL". Also, apigenin inhibited the contraction of aortic rings caused by
cumulative concentrations of calcium (0.03—3 mM) in high potassium (60 mM) medium,
with ICso of about 48 uM (Ko et al., 1991). The pretreatment by apigenin inhibited the
norepinephrine (NE, 3 uM)-induced phasic and tonic contraction in a concentration (3 5-
140 uM)-dependent manner with an IC50 of 63 uM. The conclusion was that apigenin

relaxed rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage-

and receptor- operated calcium channels (KO et al., 1991).

Singh and Handa (1995) studied the antihepatotoxic activity of A. graveolens
against paracetamol and thioacetamide intoxication in rats. The hepatoprotective effect
of methanolic extract of A. graveolens seeds on rat liver damage induced by paracetamol
(3 g kg'1 body weight) or thioacetamide (100 mg kg'1 body weight) was monitored by

several liver function tests, serum transaminases (SGOT and SGPT), alkaline

l3

 

phosphatase (SALP), sorbitol dehydrogenase (SSDH), glutamate dehydrogenase
(SGLDH) and bilirubin in serum (SBRN) as well as by assaying triglyceride and
histopathological alteration in hepatic tissues (Sing and Handa, 1995). A Significant
reduction observed in the paracetamol and thioacetamide induced increase in the levels of
SGOT, SGPT, SALP, SSDH, SGLDH, and SBRN with the pretreatment of rats with 200
mg kg'1 body weight of the methanolic extract of A. graveolens seeds were 75 and 61.9%
overall protection, respectively. Also, the histopathological studies suggested that
treatment with methanolic extract of A. graveolens reversed the hepatic lesions produced
by paracetamol as indicated from the absence of eosinophilia and presence of fewer
necrotic zones. They concluded that the Observed hepatoprotective activity of the
methanolic extract of the seeds of A. graveolens against two well known hepatotoxins,
paracetamol and thioacetamide in rats differed in their primary mechanism of inducing
hepatotoxicity. This study supported the use of A. graveolens seeds in several patented

Indian herbal preparation for liver ailments (Handa et al., 1986).

Celery juice was also studied for its hypotensive effect (Kiangsu, 1977).
Clinically, celery juice is reported to be effective in lowering blood pressure in
hypertensive patients. In this study, the celery juice was mixed with equal amounts of
honey and 40 mL of the mixture was administered orally three times a day over several

days.

Saito et al. (1976) observed antioxidative activity of petroleum ether soluble
fraction of celery seed on lard. However, Gazzani (1994) found little or no effect for

celery extract at 1-5% for anti- or pro-oxidant activities.

14

Jain and Jain (1973) studied the effect of some common essential oils on
pathogenic fungi. The steam-distilled oils from A. graveolens seeds along with some
common Indian indigenous herbs were subjected to antifungal evaluations, employing the
filter-paper-disk-diffusion-plate method (Vincent and Vincent, 1944). Celery seed oil
was tested against Trichophyton terrestre, Trichophyton tonsurans, Trichophyton
rubrum, Trichophyton mentagrophytes, Histoplasma capsulatum, Candida albicans,
Monosporium apiospermum, Beauveria sp., Philaphora verrucosa, Cryptococcus
neoformans, Microsporum mycetomi, Aspergillus flavus, Aspergillus fumigatus,
Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Sporotrichum schenckii,
Microsporum cookie, Microsporum gypseum, Epidermophyton floccosum, Nacardia
brasiliensis, F usarium oxysporum and Curvillaria lanata. Except, T. tonsurans, T.
rubrum, T. mentagrophytes, P. verrucosa, M mycetomi, S. schenckii, M. cookie and N.
brasiliensis, all the test fungi responded significantly against the celery seed essential oil.
Combination of celery seed oil with other essential oils showed synergistic antifungal
activity against all of the test fungi (Jain and Jain, 1973). This suggested that celery seed

oil can be used for the formulation of suitable topical antifungal formulations.

Phytochemical Studies

A. graveolens seed contains a wide variety of chemical constituents. The major
classes of compounds are monoterpenoids, sesquiterpene, aliphatic alcohol and carbonyl
compounds, phenols, aromatic epoxides and phthalide derivatives (Bindler and Laygel,
1986). As a class of bioactive natural products, phthalides occur widely in Umbelliferous

plants (Bjeldanes and Kim, 1977; Chulia et al., 1986; Kaouadji and Pouget, 1986a;

15

Kaouadji et al., 1984; Kaouadji et al., 1986b; Chichy et al., 1984; Banerjee et al., 1982).
Phthalides are present in celery seed oil in relatively high amounts (~7%) (Bjeldanes and
Kim, 1977) as well as in fresh celery in ppm quantities (MacLeod et al., 1988; Tang et

al., 1990).

Celery seeds were investigated for their chemical constituents as early as in 1897.
Ciamician and Silber (1897) found a sesquiterpene, selinene in celery seed oil, which was
later investigated by Schimmel Chemists (1910). The celery seed oil was fractionated
into phenols and sesquiterpenes by repeated extraction with a 5% solution of sodium
hydroxide and by distilling the separated hydrocarbon over metallic sodium. The
presence of selinene in celery seed was also confirmed by Semmler and Risse (1912),
who reported that selinene consisted of a mixture of a- and B- isomers, with B-isomer as
the predominant form. They also reported that 10 to 15% of selinene was present in

celery seed oil.

The Schimmel Chemists (1910) also reported the presence of 2.5 — 3.5% of
hydroxylated compounds in celery seed oil but did not characterize the chemistry of these
compounds. Ruzicka and Stoll (1923) found that the highest boiling fractions from
celery seed oil contained about 1% of bicyclic sesquiterpene alcohol, C15H260. Later
they came to the conclusion that selinenol obtained from selinene consisted of a mixture
of a-eudesmol and B-eudesmol. In addition, Louveau (193 7) reported that the celery seed

oil contains 2 — 3% of unidentified terpene alcohol.

Guenther (1950) noted the presence of sedanoic anhydride in celery seed Oil,
which was characterized as tetrahydro-n-butylidene phthalide. The same author reported
the sedanolide and its corresponding hydroxy acid, sedanolic acid in celery seed oil and
concluded that the sedanolide and sedanolic acid anhydride were the two constituents
mainly responsible for the characteristic odor of celery seed Oil. Limonene was identified
by the Schimmel Chemists (1910) in the early part of the twentieth century. They
reported that the celery seed oil contains about 60% of d-limonene which was further
confirmed by Guenther (1950). The presence of sedanolide in celery seed oil was
determined by Barton and De Vries (1963). 3-n-butylphthalide was reported as the main
component of celery seed oil (Barton and De Vries, 1963). Phthalides, 3-n-
butylphthalide and sedanolide were also identified as the major odor components in
celery seed (Bjeldanes and Kim, 1977). They also characterized 3-n-butyl-4,5-
dihydrophthalide (sedanenolide) for the first time as a celery-flavor compound and was
later confirmed by Uhling et al. (1987). Fehr (1979) also identified 3-n-
butylidenephthalide, 3-n-butylidene-4,5-dihydrophthalide (Z-ligustilide) and 3-
isobutylidene-3a,4-dihydrophthalide in addition to the 27 volatile components in the
essential oil from A. graveolens seeds. The celery seed oil and extracts were also
examined for their phthalide composition by Gijbels (1983) and isolated cnidilide,

neocnidilide, butylphthalide, senkyunolide and Z-ligustilide.

l7

d-limonene y-terpinene

f M

p-cymene B-myrcene

W

Ot-myrcene Ot-thujene

\

 

//

camphene oc-cedrene

 

 

Ot-pinene B-pinene

 

/

terpinolene 3-carene

20

B-eudesmol a'eUdesmOl
OH
OCH3
eugenol
OH OH
OH
p-mentha-2,8-dien-1 '01 p-mentha-8(9)-en-1,2-diol

21

HO

B-selinene elemol

 

 

 

 

caryophyllene

 

B-bisabolene Ot-bergamotene

22

The celery seed oil was analyzed by GC-MS by Masada (1976) and reported the presence
of a-pinene, camphene, B-pinene, myrcene, limonene, y-terpinene, p-cymene, octanal,
nonanal, octanol, caryophyllene and carvone. This author tentatively identified two
terpenoids, selinene and tetradecanal. One year later, Gupta and Baslas (1978) reported
the presence of carvone, piperitone, eugenol, a-pinene, terpinolene, 5-3-carene,

myrecene, terpinene and menthone in a sample of Indian celery seed oil.

carvone piperiton

menthone

23

O O
3-n-butylphthalide sedanolide
O O
O O

cnidilide neocnidilide

o l 0

O O
sedananolide Z-ligustilide

24

From the cold ether extract of the celery seeds, two dihydrofurocoumarins were
isolated using column chromatography and preparative TLC (Garg et al., 1978). These
compounds were identified by chemical and spectroscopic methods as rutaretin and its
dehydrated derivative apiumetin. In continuation of their work on A. graveolens seeds, 3
more compounds were isolated from the 320 extract (Garg et al., 1979a). These
compounds were identified as 3-methoxy-4,5-methylenedioxybenzoic acid (myristic
acid), 8-hydroxy-5-methoxypsoralen and umbelliferone. This was the first report of the
natural occurrence of myristic acid in celery seeds. In the later study, Garg et al. (1979b)
reported 6 coumarins from the petroleum ether extract of the A. graveolens seeds. Three
of these coumarins, seselin, isoimperatorin and osthenol were novel and the other two,
bergapten and isoimpinellin were known from celery seed (Musajo et al., 1954;
Innocenti, 1976). Garg and co-workers (1979c) have also isolated and characterized a
new furanocoumarin glucoside, apiumoside from the ethyl acetate extract of the celery
seeds. Garg et al. (1980) isolated and identified a new dihydrofuranocoumarin,
celereoside and isoquercitrin from the ethyl acetate extract of A. graveolens seeds. Other
natural coumarins reported were apigravin, apiumetin, apiumoside and bergapten in
celery seed (Murray et al., 1982a; Murray et al., 1982b). Ahluwalia et al. (1988) isolated
three new furanocoumarin glucosides namely, (+)-2,3-dihydro-9-hydroxy-2[1-(6-
sinapinoyl)B-D-glucosyloxy- l -methylethyl]-7H-furo[3 ,2g] [1 ]-benzopyran-7-one; (-)-
2,3,-dihydro-9-O-B-D-glucosyloxy-2-isopropenyl-7H-furo [3,2g] [1]-benzopyran-7-one;
and 5-methoxy-8-O- B-D-glucosyloxypsoralen besides previously reported celeroside and

nodakenin from the seeds of A. graveolens.

25

Forrnacek and Kubeczka (1982) examined an oil of A. graveolens using a
combination of capillary gas chromatography and l3C-NMR spectroscopy. They found
that the oil contained a-piene (0.15%), camphene (0.04%), B-pinene (0.90%), sabinene
(0.03%), 8-3-carene (0.03%), myrcene (0.79%), limonene (85.13%), B-phellandrene
(0.05%), cis-ocimene (0.01%), trans-ocimene (0.02%, p-cymene (0.18%),
pentylcyclohexadiene (0.12%), pentyl benzene (1.93%), linalool (0.09%), caryophyllene

(0.10%), B-selinene (3.68%), myristicin (0.18%) and 3-n-butylphthalide (1.93%).

 

/ \
O
O o

apigravin apiumetin

OCH3

m W \
O
O o O O O

bergapten umbelliferone

26

 

 

O
H3CO
o OCH3
a—II-I'"
seselin isopimpinellin
o /

O O

osthenol isoimperatorin

27

\ OCH3

\/ H300 o
O \/
o

apiol myristicin

   

rutaretin 8-hydroxy-5-methoxypsoralen

28

HO

0 O 0 R10 0 O

/ /
OCH3 OH
R1=Glucose
isoquercitin celereoside
O O O
\

 

nodakenin

OR
0 OH
W90
0 O
HO O O
/
R=6'-(p-coumaroyl)-B-D-glucosyl

apiumoside

29

The volatile components of celery seeds having characteristic spicy aroma and being used
as an ingredient of curry powder, were investigated by Kim et al. (1989). Steam
distillation followed by extraction with diethyl ether : n-pentane (2:1, v/v) mixture, the
volatile components, a-pinene (1.12%), myrcene (0.27%) and limonene (23.44%) were
identified by capillary GC and GC/MS. Based upon GC/MS and GC/FTIR analysis of
essential oil of celery Seed, more that 50 compounds were detected and 27 of which were
identified (Cu et al., 1990) (Table 1.1). Perineau et al. (1989) studied celery seed
essential oil and identified sixteen compounds. In addition, they also reported ligustilide

and sedanonic anhydride as components of celery oils.

In 1994, Vemin and Parkanyi analyzed the essential oils from celery seeds by
GC/MS. Among the terpene and sesquiterpene derivatives, ten compounds such as a-
thujene, (2)- or (3)-methylbutylbenzene, n-hexylcyclohexadiene, 3-pentenylbenzene, a-
cedrene, (E)-a-bergamotene, B-bisabolene, 6-selinene, octahydroazulene, Ar-curcumene
and elemol were reported as novel from celery seeds. Myrtenal, I-p-menthen-9-al (two
isomers) and 6,10,14-trimethyl-2-pentadecanone were identified as novel compounds
(Vemin and Parkanyi, 1994). The t-pinocarveol, myrtenol and verbenol were also
detected in the alcohol series (Vemin and Parkanyi, 1994). They have also confirmed the
structures of the commonly occurring phthalides present in celery seed essential oil in

addition to identifying twenty new compounds.

30

Table 1.1 The chemical composition of celery seed oil (Cu et al., 1990)

 

 

 

Compound Methods of Identification ‘ o/o
a—pinene GC/MS, GC/FTIR 1.05
camphene GC/MS trace
B—pinene GC/MS trace
sabinene GC/MS 0.76
myrcene GC/MS, GC/FTIR 0.95
6-3-carene GC/MS, GC/FTIR trace
S-phellandrene GC/MS trace
limonene GC/MS, GC/FTIR 72.16
B-phellandrene GC/MS 0.02
cis-B-ocimene GC/MS, GC/FTIR trace
trans-B-ocimene GC/MS trace
p-cymene GC/MS, GC/FTIR 0.74
pentyl benzene GC/MS, GC/FTIR 0.02
linalool GC/FTIR 1.48
isopulegone GC/FTIR 0.16
caryophyllene GC/MS, GC/FTIR 0.17
carvone GC/MS, GC/FTIR 0.09
geranyl acetate GC/MS, GC/FTIR 0.04
a-ionone GC/MS 0.05
cinnamic aldehyde GC/FTIR 0.15
thymol GC/MS 0.17
B-selinene GC/MS 12.17
a-selinene GC/MS 2.05
epoxycaryophyllene GC/MS 0.55
n-butyl phthalide GC/MS, GC/FTIR 2.56
eudesmol GC/MS 0.29
ligustilide GC/MS 2.41

 

 

 

31

 

Schmidt et al. (1995) isolated and detected a new brassinosteroid from celery seed by
GC-MS spectral analysis, which they called 2-deoxybrassinolide. Zlatanov and lvanon
(1995) investigated sterol composition of A. graveolens oil. Celery seed contains 0.6% of
sterol (Zlatanov and lvanon, 1995), in which sitosterol (27.4%) and stigmasterol have
been identified as major components. Gas liquid chromatographic analysis of celery oil
also suggested the presence of minor components, cholesterol, brassicasterol,
campesterol, A7-campesterol, AS-avenasterol, A7-stigmasterol and A7-avenasterol. The
tocopherol and tocotrienol composition of A. graveolens seed oil was also investigated

using HPLC on silica columns with fluorescence detection (Ivanov and Aitzetiiller,

1995).

Enantiomeric distributions of 3-butyl(hexahydro)phthalides in celery seed was
investigated by Bartschat et al. (1997) using enantioselective multidimensional gas
chromatography with heptakis[2,3-di-O-acetyl-6-O-(tert—butyldimethylsilyl)]-B-
cyclodextrin as the chiral stationary phase. They found 3S- configuration of 3-
butyl(hexahydro)phthalides as a dominant constituent with trace amount of 3R-
configuration of 3-butyl(hexahydro)phthalides in celery seed. Other configurations such
as 38, 3aS, 7aR; 3R, 3aR, 7aS; 3S, 3aR, 738; and 3R, 3aS, 7aR were not detectable in

celery seed.
It is clear from this review that bulk of the research on celery has focused on the

phytochemistry and bioactivities of crude extracts from it. The following chapters report

the bioassay-guided fractionation and purification of compounds possessing

32

mosquitocidal, nematicidal, antimicrobial, topoisomerase inhibitory, antiinflammatory

and antioxidant activities.

33

Chapter 2
Bioactive Compounds and 1,3-di[(cis)-9-octadecenoyl]-2~[(cis,ciS)-9,12-

octadecadienoyl] glycerol from Apium graveolens L. Seeds.*

ABSTRACT- Bioassay-directed isolation and purification of the hexane extract of
Apium graveolens L. seeds led to the characterization of three compounds, B-selinene (1),
3-n-butyl-4,5-dihydrophthalide (2) and 5-allyl-2-methoxyphenol (3). The structures of
these compounds were established by using 1H- and l3C-NMR spectral methods.
Compounds, 1-3 demonstrated 100% mortality on fourth-instar Aedes aegyptii larvae at
50, 25 and 200 ug mL“, respectively in 24 h. Also, compound 2 inhibited the growth of
Candida albicans and Candida kruseiz' at 100 ug mL". It inhibited both topoisomerase-I
and -II enzyme activities at 100 ug mL". Compound 2 displayed 100% mortality at 12.5
and 50 ug mL'l, respectively, when tested on nematodes, Panagrellus redivivus and
Caenorhabditis elegans. The triglyceride, 1,3-di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9,12-
octadecadienoyl]glycerol (4) and compound 3 were isolated for the first time from A.

graveolens seeds, although compound 4 was not biologically active.

*R. A. Momin, R. S. Ramsewak, and M. G. Nair (2000) Bioactive Compounds and 1,3-
di[(cis)—9-octadecenoyl]-2-[(cis,cis)-9,12-octadecadienoyl]glycerol from Apium

graveolens L. Seeds. J. Ag. Food Chem, 2000, 48, 3785-3788.

34

INTRODUCTION

Apium graveolens L. (Umbelliferae), celery, is a hepaxanthic herb grown as a
biennial and under certain conditions, as an annual. Celery is a native of Eurasia and is
grown mainly in coastal regions. Celery is widely cultivated in the temperate zones as an
important garden crop and the bleached leaf stalks are being relished as a popular
vegetable. Celery seeds are used in India to treat bronchitis, asthma, liver and spleen
diseases (Satyavati and Raina, 1976). A. graveolens is one of the ingredients in eight of
the thirty-three Indian polyherbal formulations with reputed life-protecting activity
(Handa et al., 1986). Several components from celery seeds were also reported to have

anticarcinogenic activity (Zheng et al., 1993).

Celery seed contains a variety of constituents. As a member of the class of
bioactive natural products, phthalides occurs widely in umbelliferous plants (Bjeldanes
and Kim, 1977; Chulia et al., 1986; Kaouadji et al., 1984; Kaouadji et al., 1986b,
Banerjee et al., 1982). Earlier studies of A. graveolens led to the isolation of limonene; p-
mentha-2,8-dien-1-ol; p-mentha-8(9)-en-1,2-diol; 3-n-butylphthalide, sedanolide (Zheng
et al., 1993); selinene (Semmler and Risse, 1912); selinenol (Ruzika and Stoll, 1923) and
seselin, isoimperatorin, osthenol, bergapten, isopimpinellin, apigravin, and apiumoside

(Grag et al., 1978; Grag et al., 1979a; Grag et al.,1979b).

Previous research on A. graveolens was focused on the isolation and structural

identification of compounds for perfumery and culinary uses. In this paper, we report,

35

three biologically active compounds (1-3) and a triglyceride (4) from the hexane extract

of celery seeds.

MATERIALS AND METHODS

General Experimental Procedures.

1H- and 13C NMR, DEPT, Arrayed Decoupling, HMQC, and HMBC spectra were
recorded on a Varian INOVA 300 MHz spectrometer. '3 C NMR and DEPT spectra were
recorded at 75 MHz. Chemical shifts were recorded in CDC]; , and the values are in 6
(ppm) based on 5 residual of CHCI3 at 7.24 for 'H NMR and CDC13 at 77 ppm for 13C
NMR . Coupling constants, J, are in Hz. The silica gel used for MPLC was Merk Silica
gel 60 (30-70 pm particle size). FAB-MS was recorded at 70eV. TLC plates (GF
Uniplate, w/binder, 250 um Analtech, Inc., Newark, DE), and preparative TLC plates
(Analtech, Sigel, 20 x 20 cm, 250, 500 um) after developing, were viewed under UV

light (254 and 366 nm). All organic solvents used were ACS reagent grade (Aldrich

Chemical Co., Inc., Milwaukee, WI).

Gas chromatography was performed using a HP 6890 gas chromatograph
(Algilent Technology, Wilmington, Delaware). Aliquots of 1 pL were injected into a
split injector at 200 OC, and separated on an HP-5 capillary column (30 In x 0.25 mm id.)
with helium carrier gas at a flow rate of 22 mL min]. Compounds were detected using

flame ionization at 250 OC. The temperature profile was 150 OC (2 min) to 200 0C (l

36

min) at 10 0C min", then to 250 0C (2 min) at 2 OC min'l. Methyl esters of palmitate,

linolenate and y-linolenate were purchased from Aldrich Chemical Co., Inc., Milwaukee,

WI.

Plant Material.

Celery seeds were provided by Asgrow Seed Company, Kalamazoo, Michigan and stored

at —20 0C until extraction.

Extraction and Isolation.

The seeds (100 g) were milled using an industrial Warring blender and extracted
sequentially with 500 mL of each hexane, EtOAc, and MeOH over a period of 48 h. The
hexane extraction afforded 17 g of residue upon removal of solvent. A portion of this
hexane extract (5 g) was stirred with acetone to yield acetone soluble (2.3 g) and
insoluble (2.6 g) portions. Fractionation of the bioactive acetone soluble portion (1.6 g)
was carried out by medium pressure liquid chromatography (MPLC) on silica gel (Sanki
Engineering Ltd., Model LBP-V pump operating at 10-15 psi; Chemco MPLC tayperling
type glass column, 55 cm in length) at 2 mL min‘1 flow rates. Fractions collected were I
(500 mL) eluted with 100% hexane, II and III with hexane: ether (10:1, 500 mL each),
IV (500 mL) with 100% acetone and V (500 mL) with 100% MeOH. The fractions I and
IV were mosquitocidal, and fraction IV was antimicrobial, nematicidal, and inhibitory to

Top-I and Top-II enzymes.

37

Fraction I was purified by repeated preparative TLC and yielded fractions A and
B. The fraction A was further purified with hexane: ether (10:1) as the mobile phase to
yield compound 4 (104 mg). Isolation of compound 1 (30 mg) from fraction B was
accomplished by using 10:1 and 50:1 hexane: ether mobile phases in repeated preparative
TLC. Fraction IV was purified by preparative TLC using hexane: ether (4:1) and yielded
four fractions. The bioactive fraction was separated into hexane soluble and insoluble
fractions. The hexane soluble fraction was further purified by preparative TLC (pentane:
ether, 4:1) and afforded a pure compound 3 (6 mg). The band at Rt=0.45 was eluted with

MeOH on a Clg-Sep Pak cartridge and yielded compound 2 (26mg).

Compound l. 1H NMR (CDCl3) 5 0.71 (s, 3H, H-l4), 0.80 (m, 1H, H-7), 1.2-1.6 [m,
10H, H-(1,2,3,6,8) x 2], 1.7 (s, 3H, H-13), 1.9 (m, 2H, H-9), 2.3 (m, 1H, H-5), 4.4-4.7
[bs,4H, H-(12,15)]; 13C NMR (CDCl3) 5 16.3 (C-14); 21.0 (C-l3); 23.5, 26.8, 29.5, 36.9,
41.2, 41.9 (C-1,2,3,6,8,9); 36.0 (C-lO); 45.9, 49.9 (C-5,7); 105.4, 108.1 (C-12,15); 150.8,
151.0 (C-4,11). 1H NMR data of compound 1 were found to be in agreement with

previously published data (Bowden et al., 1978).

Compound 2. 1H NMR(CDC13) 8 0.9 (t, 3H, J=7.2, H-4’), 1.2-1.8 [m, 6H, H-(l ’,2’,3’)],
2.45 (m, 4H, H-4,5), 4.9 (m,.1H, H-3), 5.9 (m, 1H, H-6), 6.2 (d, 1H, J=10, H-7); l3C
NMR (CDC13) 6 13.8-22.4 (C-l’,2’,3’,4’); 26.7-31.8 (C-4, 5); 82.5 (C-3); 116.8 (C-7);
128.3 (C-6); 124.5-135 (C-8, 9); 161.4 (C-l). The spectral data confirmed that

compound 2 is 3-n-butyl-4,5-dihydrophthalide (Bjeldanes and Kim, 1977).

38

Compound 3. 1H NMR (CDCl3) 5 3.32 (d, 2H, J=7.0, H-7), 3.84 (s, 3H, -OCH3), 5.08
(m, 2H, H-9), 5.45 (s, 1H, -OH), 5.90 (m, 1H, H-8), 6.66-6.69 (m, 2H, H-3,5), 6.83 (d,
1H, J=8.5, H-6); ”C NMR (CDCI3) 8 39.8 (C-7), 55.8 (-OCH3), 110.9 (C-9), 114.0 (C-
8), 115.4 (C-6), 121.1 (C-5), 131.7 (C-4), 137.8 (C-3), 146.3 (C-2), 146.4 (C-l). The 1H
NMR data were found to be in agreement with previously published data for compound 3

(Kurihara and Kikuchi, 1979).

Compound 4. '11 NMR (CDC13) 6 0.86 [t, 9H, J=6.6, H-(18’ x 2), 18”], 1.24 [m, 54H, H-
(4’-7’,12’-17’) x 2 and H-(4”-7”, 15”-17”)], 1.6 [m, 6H, H-(3’ x 2), 3”], 2.02 [m, 12H, H-
(8’, 11’) x 2 and H-(8”,14”)], 2.3 [1, 6H, J=7.2, H-(2’ x 2), 2”], 2.76 [dd, 2H, J=6.1, 5.5,
11-11”], 4.12 (dd, 211, J=18.0, 6.0, H-la, 3a), 4.34 (dd, 2H, J=16.2, 4.2, H-lb, 3b), 5.3—5.4
[m, 9H, H-(9’, 10’) x 2. H-(9”,10”,12”,13”) and H-Z]; 13C NMR (CDCl;;) 8 14.0 (018”);
14.1 (C-18’ x 2); 22.6, 22.7, 24.4, 24.8, 25.6, 26.8, 27.2, 27.3, 29.1-29.7 [C-(4’- 8’, 11’-
17’) x 2, 4”-8”, 14”-17”]; 31.5 (C-3”); 31.9 (C-3’ x 2); 33.9 (C-2’ x 2); 34.0 (C-ll”);
34.2 (C-2”); 62.1 (C-1, 3); 68.8 (C-2); 127.9 — 130.5 [C-(9’, 10’) x2, C-
(9”,10”,12”,13”)]; 172.8 (C-l”); 173.2 (01’ x 2). The 'H- and ”C- NMR data for

compound 4 were identical to the published finding of Chandra and Nair (I993).

Saponification and Methylation of compound 4 and standards.

Compound 4 (7 mg) was stirred with 5% NaOH in MeOH (1 mL) for 5 min followed by

acidification with 6N HCl in MeOH. This solution containing free fatty acids was then

dried under a stream of nitrogen. Diazomethane was prepared by reacting N-nitroso-N-

39

methylurea with concentrated KOH solution in ether (Kelm and Nair, 1998). As the
diazomethane product formed, it dissolved into the organic ether phase. This yellow
ether solution containing the diazomethane product was collected and used to methylate
the free fatty acids obtained in the previous step. Also, the diazomethane solution was
used to methylate oleic, stearic and linoleic a'cids separately. The methylated products

were dissolved in hexane and filtered to remove any solids prior to GC analysis.

Mosquitocidal Assay.

A. aegyptii larvae were reared in our laboratory from eggs. Eggs were hatched in 500 mL
of distilled, degassed water prepared by sonication (30 min) and larvae were fed with
approximately 5 mg of bovine liver powder. Ten to fifteen 4Lh instar larvae were placed
in 980 uL of distilled water in test tubes and 20 uL of DMSO or DMSO solutions
containing the appropriate concentration of test compounds were added and left at room
temperature. Initially, the crude extracts and pure compounds were tested at 250 and
100 ppm concentration, respectively. The test concentrations for pure compounds were
then serially diluted to 0.1 ppm as the final concentration. Pure DMSO was used as
solvent control. The assays were conducted in triplicate, and the numbers of dead larvae

were recorded at 2,4, 12, and 24 h intervals (Roth et al., 1998; Ramsewak et al., 1999b).

Nematicidal Assay.

The nematode cultures, C. elegans and P. redivivus were maintained in our laboratory.

40

C. elegans was grown on NG agar media containing a strain of Escherichia coli in
disposable petri dishes wet with 2-4 mL of physiological saline solution. P. redivivus
was maintained in axenic, liquid Basal Heme media (5 mL) in scintillation vials. The
cultures were stored at room temperature and subcultured prior to the assay. The assay
was conducted in Corning polystyrene 96 well plates. The nematodes were added to 1
mL of physiological saline solution in a scintillation vial. This solution was diluted until
the nematodes count were 15-20 in a 48 11L aliquot. Forty eight uL of solution
containing nematodes was delivered to each of three wells per treatment. Two uL of
DMSO (50%) or DMSO (50%) and test compounds were added to each well. The plate
was covered, parafilmed, and kept in a humid chamber. The number of dead nematodes

was recorded every at 2, 4, 6, 8, and 24 h by observing under a microscope (Nair et al.,

1989).

Antimicrobial and Topoisomerase-I and -II Inhibitory Assays.

The test organisms Candida kruseii (MSU strain) and Candida albicans (MSU strain)
used for antimicrobial bioassays were cultured in Petri dishes containing YMG media (20
mL) and Saccharomyces cerevisiae mutant cell cultures, JN 394, JN 394 t -1, and JN 394 t -
2-5, used for the topoisomerase assay, were cultured in Petri dishes containing YPDA
medium (20 mL). Physiological saline solution (2-3 mL) was added to fully grown plate
of each organism and then suspensions were diluted to obtain 5 x 106CFU/mL. A 50 uL
aliquot of this suspension was then used to inoculate culture tubes containing the

corresponding media (930 11L). DMSO or test compounds dissolved in DMSO were

41

added to the inoculated tubes (20 uL),at concentrations ranging from 100 to 0.1 ppm.
The tubes containing cell cultures and compounds were incubated at 27 0C on a rotary
orbital shaker at 120 rpm for 72 h. MICmo values for the test compounds were recorded
for each test organism at the end of incubation period (Chang et al., 1995; Roth et al.,

1998; Ramsewak et al., 1999b).

RESULTS AND DISCUSSION

Seeds of A. graveolens were extracted with hexane, and by successive silica gel MPLC,
and preparative TLC gave four compounds, 1-4. The hexane extract was fractionated
into acetone soluble and insoluble fractions. The bioactive acetone soluble fraction was
subjected to medium-pressure liquid chromatography. Fraction A eluting with 100%
hexane and fraction D eluting with hexane: ether (10:1) was found to be active. Fraction
A was purified by repeated preparative TLC to yield 1,3-Di[(cis)-9—octadecenoyl]-2-
[(cis,cis)-9,12—Octadecadienoyl]glycerol (4) and B-selinene (l). The repeated preparative
TLC of fraction D yielded bioactive compounds 3-n-butyl 4,5-dihydrophthalide (2) and

5-allyl-2-methoxyphenol (3).

1H- and l3C-NMR and DEPT spectral data were used to determine the structure of
compound 1. Singlets at 1.7 and 0.71 ppm , each integrated for 3 protons, indicated the
presence of two methyl groups (C-13, C-14) attached to olefinic and quaternary carbons,
respectively. The broad singlet at 4.4-4.7 ppm indicated the presence of vinyl protons at

C-12 and C-15 in support to presence of '3C NMR signals at 105.4 and 108.1 ppm. The

42

DEPT spectrum supported the lH NMR and '3 C NMR data. The IH NMR spectrum of 1

was identical to the published finding of Bowden et al., (1978).

 

The structure of compound 2 was determined by using lH, '3 C NMR, and DEPT
spectral data. The two-dimensional HMQC proton-carbon correlations, HMBC proton-
carbon long range coupling and arrayed-decoupling experiments facilitated further
evidence to support the 1D NMR experiments. The lH NMR spectrum displays a doublet
at 6.2 ppm (1H, J=10 Hz) and a multiplet at 5.9 ppm (1H) for the vinyl protons, H-7 and
H-6, respectively; as well as a multiplet at 4.9 ppm for H-3. The 1H NMR spectral data of

compound 2 were identical to previously published data (Bjeldanes and Kim, 1977).

 

43

The structure of compound 3 was determined by using IH, 13 C NMR, and DEPT
spectral data. A singlet at 3.84 ppm corresponded to methoxy protons. A singlet at 5.45
ppm was determined to be hydroxy group as indicated by the disappearance of this peak
following a D20 shake in the lH NMR spectrum. Presence of two peaks, at aromatic
region (6.66—6.83 ppm), integrated for 3 protons, indicated a trisubstituted aromatic ring.

This proton data were found to be in agreement with those previously published

(Kurihara and Kikuchi, 1979).

 

The structure of compound 4 was determined by using 1H and '3 C NMR, and MS
Spectroscopy. The 1H NMR signals at 5 4.12 and 4.34 corresponded to 2 x CH2-
methylene protons of a glycerol backbone. The overlapping multiplets at 8 5.3 for 8
protons correlated with unsaturated carbons at 6 127.9-130.5 indicated the presence of 4
double bonds in this molecule. MS peaks at m/z 883 [C57H10206 +H+], 617 [C57H10206-
C13H310]+, 263 [C13H310]+, 265 [C13H33O]+ supported the presence of oleic and linoleic
acids. The GC profile of methylated hydrolysis product of this molecule confirmed the
presence of the methyl esters of oleic and linoleic acids with a ratio of 2:1, respectively.

The retention times for both methyl esters were identical to those of authentic samples of

44

oleic and linoleic acid methyl esters analyzed under the same conditions. The spectral

data of compound 4 were identical to the published finding by Chandra and Nair (1993).

 

Mosquitocidal assays using Aedes aegyptii on compounds 1-4 indicated that
compounds 1, 2 and 3 were active. Compound 1, 2 and 3 had an LDloo (24 h) at 50, 25
and 200 pg mL", respectively (Fig. 2.1). Compound 2 displayed nematicidal activity on
P. redivivus and C. elegans. LDmo values in 24 h were 12.5 and 50 ug mL'lfor P.
redivivus and C. elegans, respectively (Fig. 2.2). Compound 2 showed topoisomerase-I
and -II inhibitory activity when tested on S. cerevisiae mutant strains. JN394 is
hypersensitive to both topoisomerase-I and -——II poisons, while JN394 t-1 lacks the top-I
gene and therefore shows a lack of response to topoisomerase-I poisons. JN394 t-2-5
carries the top-11 gene, which is resistant to topoisomerase-II poisons but responds to
topoisomerase-I poisons. MICloo determination of the compound 3 indicated that it

completely inhibited topoisomerase-I and II activity at 100 ug mL'l concentration. The

zone of inhibition at 50 pg mL’l concentration of compound 2 on YPDA plate were 1.1,

45

1.1 and 1.2 cm for JN394, JN394 L1 and JN394 t_2_5, respectively. Antimicrobial assays
showed that compound 2 was active when tested on C. albicans and C. kruseii (MSU
strains). The inhibition zone for C. albicans and C. kruseii was found to be 1.1 and 1.0

cm at the concentration of 50 pg mL", respectively. Compound 2 had an MICloo at 100

ug mL'l concentration.

Figure 2.1. Percent mortality of 4‘h instar A. aegyptii larvae for compounds 1-3 at
24 h. Statistical analysis was done using ANOVA (p 5 0.01) and the
means were compared by calculating least significant difference

(LSD).

100 ICcntrol
ICompound-3
1 UCompound-t
ICompound-z
ao .
so 1
4o .
20 l
-
-

100 v 7777‘ THAT?“
50 25 , N H T WIT-77

‘lo Mortality

 

 

200

Concentration (pg mL")

The flavor of celery seed oil is mainly due to B-selinene and phthalides (Lund,

1978). Terpenes, terpenoids and phthalides have been implicated as being natural

46

insecticides, insect pheromones, mammalian sex hormones and they have been used since
antiquity as ingredients of flavors, preservatives, perfumes, medicines, narcotics and
pigments. The present study supports that the observed biological activity of celery seeds
is part due to the presence of terpenes and phthalides. To the best of our knowledge, this
is the first report of compounds 3 and 4 from A. graveolens seeds, although compound 4
is not biologically actiVe. Till now, there are no reports on the biological activity of

compounds 1-3 although compound 2 was used as one of the component of perfume.

Figure 2.2. Percent mortality of C. elegans and P. redivivus nematodes for
compound 2 at 24 h. Statistical analysis was done using ANOVA
(p _<_ 0.01) and the means were compared by calculating least

significant difference (LSD).

 

 

 

 

 

IControl
100. l W T IC. elegans
so I DP. red/Vlvus
l
:E 60
g 1
=2 404
20 - L
l L g
0 ~ _ - - .Q Q/
100 50 25 l -/
12.5 6.25 1 701» SJ
Concentration (119 mL") ‘ 0

47

Chapter 3

Mosquitocidal, Nematicidal and Antimicrobial compounds from Apium

graveolens L. seeds*

ABSTRACT- The methanolic extract of Apium graveolens seeds was investigated for
bioactive compounds. Mosquitocidal, nematicidal and antimicrobial compounds,
sedanolide (5), senkyunolide-N (6) and senkyunolide-J (7) were isolated and
characterized from A. graveolens seeds. Their structures were determined by 1H and '3 C
NMR spectral methods. Compounds 5-7 gave 100% mortality at 25, 100 and 100 ug mL'
', respectively, on the nematode, Panagrellus redivivus. Compound 5 showed 100%
mortality at 50 ug mL'l on nematode, Caenorhabditis elegans, and fourth-instar mosquito
larvae, Aedes aegyptii. Also, it inhibited the growth of Candida albicans and Candida
parapsilasis at 100 ug mL". Compounds 6 and 7 were isolated for the first time from A.
graveolens. This is the first report of the mosquitocidal, nematicidal and antimicrobial

activities of compounds 5-7.

*R. A. Momin and M. G. Nair (2000) Mosquitocidal, nematicidal and antimicrobial

compounds from Apium graveolens seeds. J. Ag. Food Chem, Submitted, August, 2000.

48

INTRODUCTION

Apium graveolens L., celery, is widely grown in the temperate zone as a garden crop and
its leaf stalks are used as a popular vegetable. In the USA, celery fruit, commonly known
as “celery seed”, was largely imported from Europe but since World War-II, it has been
supplied from domestic growers, especially in Michigan and Wisconsin. Celery seeds
possess a characteristic aroma and pungent taste and are used as a condiment in the
flavoring of food products. Celery can be classed both as a seasoning and as a vegetable.
For seasoning purpose, the seeds and the plant, as well as the essential oil distilled from
the seeds are used (Salzer, 1975). Celery seed extracts are extensively used as flavoring
ingredients in many food products, including alcoholic and nonalcoholic beverages,
frozen dairy desserts, candy, baked goods, gelatins, puddings, meat products, condiments
and relishes, soups, gravies, snack food, and others. Celery seeds are used for treating

bronchitis, asthma, liver and spleen diseases (Chopra et al., 1958; Satyavati and Raina,

1976)

Sedanolide, considered to be one of the celery flavor compounds (Bjeldanes and
Kim, 1977; Tang et al., 1990) was isolated and identified from celery seed oil in 1950
(Guenther, 1950). Since the isolation of sedanolide, several other phthalides such as
sedanenolide, 3-n-butylphthalide, cnidilide, neocnidilide, ligustilide and 3-isobutylidene-
3a,4-dihydrophthalide were reported from celery seed oil (Bjeldanes and Kim, 1977;
Fehr, 1974; Uhling et al., 1987). In this paper we report for the first time the

mosquitocidal, nematicidal and antimicrobial activities of compounds 5-7.

49

MATERIALS AND METHODS

General Experimental Procedures.

All NMR spectra (1H and '3 C) were recorded on a Varian INOVA 300 MHz
spectrometer. '3 C NMR spectra were recorded at 75 MHz. Chemical shifts were
recorded in CDC13, and the values are in 6 (ppm) based on 8 residual of CHCl3 at 7.24 for
1H NMR and CDCI3 at 77 ppm for 13C NMR. Coupling constants, J, are in Hz. The
silica gel used for VLC and MPLC was Merk Silica gel 60 (30-70 urn particle size). For
preparative HPLC (LC-20, Japan Analytical Industry Co., Tokyo) purification, two
JAIGEL-ODS, A-343-10 (20 mm x 250 mm, 10 um, Dychrom, Santa Clara, CA)
columns were used in tandem. Peaks were detected using a UV detector equipped with
model D-2500 Chromato-integrator (Hitachi, Tokyo). The CD analysis of compounds 6
and 7 were performed on JASCO J-710 CD-ORD spectropolarimeter. Nitrogen was
generated by a nitrogen generator model NG-150 at a rate of 40 L min". Compounds 6
and 7 were dissolved in methanol separately, and the CD was determined at 185-400 nm.
All solvents were ACS reagent grade and were purchased from Aldrich Chemical Co.,

Inc.

Plant material

Celery seeds were donated by Asgrow Seed Company, Kalamazoo, Michigan and stored

at —20 0C until extraction.

50

Extraction and isolation

The seeds (905 g) were milled using an industrial Warring blender and extracted with
hexane (4 x 1.5 L, 48 h) to yield the hexane extract (85 g). The residue was then
extracted with MeOH (3 x 1.5 L, 48 h) to yield MeOH extract (20 g). The solvents were
evaporated under reduced pressure at 40 0C to yield crude extracts. The MeOH extract
(19 g) was stirred with hexane (2 x 400 mL, 15 min) to yield hexane soluble (7.4 g) and
insoluble (11.5 g) fractions. The hexane soluble fraction (6 g) was further separated by
VLC followed by MPLC on silica gel (Sanki Engineering Ltd., Model LBP-V pump
Operating at 10-15 psi; Chemco MPLC tayperling type glass column, 55 cm in length)
using hexane with increasing amount of acetone and finally with MeOH as the eluting
solvents. The fractions collected were: A (404 mg, hexane-acetone, 4:1, 250 mL); B (142
mg, hexane-acetone, 2:1, 80 mL); C (483 mg, hexane acetone, 2:1, 100 mL); D (79 mg,
hexane-acetone, 2:1, 120 mL); E (61 mg, hexane-acetone, 1:1, 100 mL); F (89 mg,
hexane acetone, 1:1, 200 mL); G (35 mg, hexane-acetone, 1:1, 100 mL); H (14 mg, 100%
acetone, 20 mL); 1 (33 mg, 100% acetone, 130 mL), and J (8 mg, 100% MeOH, 250 mL).
Mosquitocidal, nematicidal and antimicrobial bioassays on these fractions revealed that
fraction B was active. The bioactive fraction B (135 mg) was purified by preparative
TLC (hexane-acetone, 20:1 x 3) to yield three major bands: I (48 mg, Rf 0.18); 11 (65 mg,
Rf 0.35) and III (16 mg, Rf 0.50). Band II was biologically active and was further
purified by repeated preparative TLCs (hexane-ether 8:1 x 4 and 2:1 x 1) to yield a pure

compound 5 (22 mg).

51

The hexane insoluble fraction (11 g) of MeOH extract was separated into
chloroform soluble (2.1 g) and insoluble (8.2 g) fractions by stirring it with chloroform (2
x 50 mL, 15 min). The chloroform soluble fraction (2.1 g) was further separated into
hexane soluble (607 mg) and insoluble (1.5 g) fractions. The hexane insoluble fraction
was further separated into methanol-water, 75:25 soluble and insoluble fractions and
precipitates were removed by centrifugation. The soluble fraction (684 mg) was
separated into 8 fractions by preparative HPLC using methanol-water, 75:25 as a mobile
phase at flow rate of 3 mL min”. Fraction II (R; = 48 min, 75 mg) was further purified
by HPLC using methanol-water, 60:40 as a mobile phase at flow rate of 2 mL min'1 to
yield 1:1 mixture of compounds 6 and 7 (R. = 65 min, 12 mg). This mixture was
separated in to pure compounds 6 and 7 by HPLC using H20-THF, 90:10 as a mobile

phase at the flow rate of 4 mL min".

Compound 5. 1H-NMR (300 MHz, CDCI3): 5 0.83 (3H, t, J=7.1 Hz, H-1 1), 1.00-2.20
(10H, m, H-4,5,8,9,10), 2.22 (1H, m, H-3a), 2.41 (2H, m, H-6), 3.87 (1H, m, H-3), 6.66
(1H, dd, J=6.3, 3.0 Hz, 11-7); 13C-NMR (75 MHz, CDC13): a 13.87 (C-1 1), 20.74 (C-S),
22.50 (C-10), 24.96 (C-4), 25.34 (C-3a), 27.49 (C-8), 34.30 (C-9), 43.04 (C-6), 85.35 (C-
3), 131.11 (C-7a), 135.20 (C-7), 170.24 (C-l). Compound 5 was identified as sedanolide.
The spectral data of compound 5 were identical to the published values for sedanolide

(Zheng et al., 1993).

Compound 6. 1H-NMR (300 MHz, CDC13): 5 0.91 (3H, t, J=7.2 Hz, H-1 1), 1.30-1.45
(4H, m, H-9,10), 1.50-2.21 (4H, m, H-5,8), 2.35-2.40 (2H, m, H-4), 3.91 (1H, ddd, J=9.8,

6.1, 3.3 Hz, H-6), 4.41 (1H, dddd, J=6.1, 2.5, 2.0, 1.9 Hz, H-7), 4.86 (1H, ddd, J=8.1, 3.6,

52

2.4 Hz, H-3); l3C-NMR (75 MHz, CDC13): 8 13.80 (C-1 1), 21.3 (C-4), 22.4 (C-10), 26.5
(C-5), 26.8 (C-9), 31.9 (C-8), 67.4 (C-7), 71.5 (C-6), 82.9 (C-3), 126.3 (C-7a), 166.7 (C-
3a), 173.0 (C-1). Compound 6 was identified as senkyunolide-N by comparison of its lH-

and 13C- NMR spectral data with published values (Naito et al., 1992).

Compound 7. IH-NMR (300 MHz, CDC13): 8 0.91 (3H, t, J=7.2 Hz, H-1 1), 1.30-1.45
(4H, m, H-9,10), 1.50-2.21 (4H, m, H-5,8), 2.35-2.40 (2H, m, H-4), 3.91 (1H, ddd, J=9.8,
6.1, 3.3 Hz, H-6), 4.41 (1H, dddd, J=6.1, 2.5, 2.0, 1.9 Hz, H-7), 4.86 (1H, ddd, J=8.1, 3.6,
2.4 Hz, H-3); l3C-NMR (75 MHz, CDC13): 6 13.80 (C-1 1), 21.] (C-4), 22.3 (C-IO), 26.3
(C-5), 26.6 (C-9), 31.8 (C-8), 67.3 (C-7), 71.3 (C-6), 82.8 (C-3), 126.1 (C-7a), 166.5 (C-
3a), 172.9 (C-l). Compound 7 was identified as senkyunolide-J by comparing its 1H- and

13C- NMR spectral data with published values (Naito et al., 1992).

Mosquitocidal Assay.

Fourth instar mosquito larvae, A. aegyptii, were reared from neonates in our laboratory.
Ten to 15 larvae were placed in 980 uL of degassed distilled water and 20 uL of DMSO
or DMSO solution containing test extracts or pure compounds were added. Extracts were
tested at 250 ug mL'1 and pure compounds were tested at a 1-200 ug mL'l concentrations.
There were three replicates per treatment. The numbers of dead larvae were recorded at
2-, 4-, 6- and 24- h intervals. The control was prepared with 980 uL of degassed distilled

water and 20 11L of DMSO solution to which larvae were added (Roth et al., 1998).

53

Nematicidal Assay.

The nematode cultures, P. redivivus and C. elegans were maintained in our laboratory. P.
redivivus was grown in axenic, liquid Basal heme media (5 mL) in scintillation vials. C.
elegans was maintained on NG agar media containing 3 strains of Escherichia coli in
disposable petri dishes wet with 2-4 mL of physiological saline solution. The cultures
were stored at room temperature and sub-cultured prior to the assay. The assay was
conducted in Corning polystyrene 96-well plates. The nematodes were added to 1 mL of
physiological saline solution in a scintillation vial. This solution was diluted until the
nematodes count were 15-20 in a 48 11L aliquot. This solution (48 uL) containing
nematodes was delivered to each of three wells per treatment. Two 11L of DMSO (50%)
and test compounds were added to each well. The plate was covered, parafilmed, and
kept in a humid chamber. The number of dead nematodes recorded every at 2-, 4-, 6-, 8-

and 24- h by observing under a microscope (Nair et al., 1989).

Antimicrobial Assay.

Compounds 5-7 were evaluated for antimicrobial activities according to the reported
procedure (Chang et al., 1995). The test organisms, C. albicans (MSU strain) and C.
parapsilasis (MSU strain) used for antimicrobial bioassays were cultured in petri dishes
containing YMG media (20 mL). Two to three mL of physiological saline solution was
added to fully grown plate of each organism and then suspensions were diluted to obtain
5 x 106 CPU mL'l. Bioassays were conducted by spreading 50 uL of the desired cell

suspension on petri dishes of the YMG media. DMSO or test compound dissolved in

54

“5:332:11.

 

DMSO (20 11L) were spotted carefully on the bioassay plates at varying concentrations.
The plates were allowed to dry in a laminar flow hood and then incubated at 27 0C for 72
h. The zone of inhibition was measured in mm (Chang et al., 1995, Roth et al., 1998).
Minimum inhibitory concentration (MlCmo) Was determined for compounds 5-7

according to the published procedure (Nair et al., 1989).

RESULTS AND DISCUSSION

Celery seeds were milled and extracted sequentially with hexane and MeOH. MeOH
extract was partitioned into hexane soluble and insoluble fractions. Preliminary
bioassays indicated that hexane soluble fraction was active on C. albicans, C.
parapsilasis, mosquito larvae, A. aegyptii, and nematodes, C. elegans and P. redivivus,
where as hexane insoluble fraction was active on nematode, P. redivivus. The hexane
soluble fraction was purified by VLC and MPLC on silica gel using hexane with
increasing amount of acetone and finally with MeOH as the eluting solvents. Ten
fractions, A-J, were collected and tested for nematicidal activity. The active fraction B,
eluting with hexane-acetone was further purified by repeated preparative TLC to yield

compound 5.

The hexane insoluble fraction of the MeOH extract of celery seed was further
partitioned to yield chloroform soluble and insoluble fractions. The chloroform soluble
fraction was active against P. redivivus. This fraction was further fractionated in to
hexane soluble and insoluble fractions. The hexane insoluble fraction was dissolved in

MeOH and precipitated with water and centrifuged. Purification of the MeOH-H20

55

soluble fraction was carried out by HPLC on two JAIGEL-ODS columns using MeOH-
H20. The fraction II (R, = 48 min) was active on P. redivivus and was further purified by
HPLC using MeOH-HZO as the mobile phase to yield a mixture of compounds 6 and 7
(R. = 65 min, 12.5 mg). This mixture was separated into compounds 6 and 7 by HPLC

using HzO-THF as the mobile phase.

The structure of compound 5 was confirmed by 1H- and 13C- NMR spectral data

and was identical to the published spectral data for sedanolide (Zheng et al., 1993).

Compounds 6 and 7 were identified as senkyunolide-N and senkyunolide-J,
respectively, by comparison of their 1H- and '3C- NMR spectral data to that of the
published data (Naito et al., 1992). The CD spectrum (Fig. 3.1) of compound 6 showed a
positive Cotton effect at 194 nm indicating that compound 6 has the 6S and 7S
configuration, while compound 7 showed a negative Cotton effect at 194 nm indicating
that 7 has 6R and 7R configuration (N aito et al., 1992). Thus, the structural identities and
stereochemistry of Compounds 6 and 7 were confirmed as senkyunolide-N and

senkyunolide-J (N aito et al., 1992). Compounds 6 and 7 were previously reported from

56

 

40

 

Compound 6

 

 

 

 

 

 

 

 

 

CD
[61 3 _
Compound 7
194 nm
-30 i- l l l l
1 80 200 250 300
Wavelength[nm]

Figure 3.1. CD Spectra of compounds 6 and 7 in MeOH

OH\\\“

 

OH

57

the dried rhizomes of Ligusticum chuangxions (N aito et al., 1992). 'This is the first report

of the isolation of compounds 6 and 7 from A. graveolens.

Compound 5 exhibited 100% mortality (LDIOO) at 25 and 50 pg mL'1 when tested
on P. redivivus and C. elegans, respectively, (Fig. 3.2 and 3.3). At 100 pg mL'l,
compounds 6 and 7 showed 100% mortality when tested on P. redivivus (Fig. 3.2). Also,
compound 5 gave 100% mortality at 50 pg mL'1 when tested on fourth instar A. aegyptii
larvae. In the preliminary bioassay, compound 5 gave a zone of inhibition of 11 mm each
for C. albicans and C. parapsilasis at 100 pg mL'l on YMG culture plates. Also, it
completely inhibited the growth of C. albicans and C. parapsilasis at 100 pg mL".
Compounds 5-7 were showed no activity when tested against bacteria, Escherichia coli,

Staphylococcus epidermidis and Streptococcus aureus (Chang et al., 1995).

Phthalides are bioactive natural products and occur widely in umbelliferous
plants. Besides its role in the characteristic odor of celery, sedanolide was previously
reported as tumor inhibitor in forestomach of female A/J mice by inducing glutathione S-
transferase enzyme activity (Zheng et al. 1993). The Chinese traditional plant, L.
chuangxiong has been used to treat headache, anemia, feeling of cold and irregularity of
menstruation. Senkyunolide-N and —J were reported from the rhizomes of L.
chuangxiong (Naito et al., 1992). These results support the notion that phthalides are
biologically active. The five-member lactone ring along with butyl side chain in
phthalides may be important for observed biological activities. Zheng et al. (1993)
reported that the five member lactone ring in phthalides is important for the high

glutathione S-transferase activity. Celery seed extracts and oil which are used in

58

s

perfumery, for flavoring, seasoning and pharmaceutical preparation have the potential to
be used as dietary supplements, if toxicity and side effects are minimal. The nematicidal

and mosquitocidal activity of compounds 5-7 suggest that further examination is required

for their potential toxicity and side effect.

Figure 3.2. Percent nematicidal activity against P. redivivus for compounds 5-7 at
24 h. Statistical analysis was done using ANOVA (p 5 0.01) and the

means were compared by calculating least significant difference

(LSD).

I Control

I Compound-7
D Compound-6
I Compou nd-5

% Mortality

 

Q Compound-6
‘ - . /
12.5 -/ Control
1 0
Concentration (pg mL")

59

Figure 3.3.

% Mortality

Percent nematicidal activity against C. elegans for compound 5 at
24 h. Statistical analysis was done using ANOVA (p _<_ 0.01) and the

means were compared by calculating least significant difference

(LSD).

. COMI'Ol
Cl Compound-5

 

 

 

 

orgy}
20° Th" 0 a - Carpomd-s

1 7 wNN
25 “oh—m-
12,5 625 $5 Control

Concentration (pg mL") 0

60

 

Chapter 4

Antioxidant, Cyclooxygenase and Topoisomerase Inhibitory compounds

from Apium graveolens Linn. Seeds.*

ABSTRACT— Cyclooxygenase inhibitory and antioxidant bioassay-directed extraction
and purification of the celery seeds yielded compounds; sedanolide [5], senkyunolide-N
[6], senkyunolide-J [7], 3-hydroxymethyl-6-methoxy-2,3-dihydro-lH-indol-2-ol [8], L-
tryptophan [9], and 7-[3-(3,4-dihydroxy-4-hydroxymethyl-tetrahydro-furan-2-yloxy)-4,5-
dihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy]-5-hydroxy-2-(4-hydroxy-3-
methoxy-phenyl)-chromen-4-one [l0]. Compounds 5-7 were isolated and characterized
according to procedures described in Chapter 3. The structures Of compounds 5-10 were
established using spectroscopic methods. Isolation and identification of compound 8 from
celery seeds is for the first time and compound 8 is a novel natural product. At 250 pg
mL", compounds 5-10 displayed 24, 32, 36, 52, 31 and 29 % of prostaglandin H
endoperoxide synthase-I (COX-1) and 24, 29,25, 40, 32 and 21 % of prostaglandin H
endoperoxide synthase-II (COX-II) inhibitory activities at pH 7, respectively. The
acetylated product (11) of compound 8 also inhibited the COX-1 and COX-II enzymes at
31 and 28 %, respectively, when tested at 250 pg mL'I concentration. The COX-1 and
COX-II enzyme inhibitory activities of compounds 5-10 were also studied at different
concentrations. Compounds 9 and 10 exhibited good antioxidant activity at 125 and 250
pg mL'l concentrations. Compounds 5-7 exhibited topoisomerase-I and —II enzyme
inhibitory activity at 100, 200 and 200 pg mL'l concentrations, respectively.

*R. A. Momin and M. G. Nair (2000) Antioxidant, Cyclooxygenase and Topoisomerase

Inhibitory compounds from Apium graveolens Linn. Seeds. Will be submitted to J. Nat. Prod.

61

INTRODUCTION

A review of the literature indicates that celery has been cultivated for the last 3000 years,
notably in pharaonic Egypt, and it was known in China in the 5th century BC (Chevallier,
1998). Throughout history, celery has been used as a food, _and at various times both the

whole plant and the seeds have been taken medicinally.

The characteristic odor of celery essential oil is due to a series of phthalide
derivatives (Bjeldanes and Kim, 1977). Sedanolide, sedanonic anhydride, 3-n-butyl
phthalide, and other minor phthalides are reported to be the major constituent of celery
seed oil (Lund, 1978). Celery seed or celery seed extracts are used as flavoring for
preparing herbal combinations sold as dietary supplements and also in antirheumatic
formulations. Celery seeds are also used specifically for arthritic pain relief (Chevallier,
1998). The seeds also have a reputation as a carrninative with a mild tranquillizing effect
(Chevallier, 1998). Today, celery seeds are used for treating rheumatic conditions and
gout. They help the kidneys dispose of urates and other unwanted waste products, as well
as working to reduce acidity in the body as a whole (Chevallier, 1998; Boulos, 1983).
The seeds are useful in detoxifying the body and improving the circulation of blood to the
muscles and joints (Houghton and Manby, 1985). Celery seeds have diuretic and
antiseptic action (Boulos, 1983; Houghton and Manby, 1985; Chevallier, 1998). They are
an effective treatment for cystitis, and to disinfect the bladder and urinary tubules. Celery
seeds are beneficial for chest problems such as asthma, bronchitis and when used in
combination with other herbs, help to reduce blood pressure (Chevallier, 1998; Styavati

and Raina, 1976; Kiangsu, 1977). Some nitrogenous and non-nitrogenous portions of the

62

celery seed essential oil were separated and tested for their effects on central nervous
system (CNS) (Kulshrestha et al., 1967; Kulshrestha et al., 1970; Satyavati and Raina,
1976). Only the nitrogenous part was found to have CNS activity. It provided protection
against supramaximal seizure threshold test and has tranquillizing activity (Kulshrestha et
al., 1967). Although celery seeds are often used in preparations for treating rheumatism,
arthritis and as a sedative and spasmolytic, the German Federal Health Office’s
Commission E monograph does not recommend its use because of insignificant scientific
proof of efficacy (Houghton and Manby, 1985). A. graveolens was assessed for its
antiinflammatory activity on intact rats by measuring the suppression of carrgeenan-
induced paw edema (Al-Hindawi et al., 1989). However, any link between known

constituents in celery with the observed antiinflammatory activity was not established.

Much of the impetus for research on A. graveolens has been due to its role in
traditional folk medicine. Most of these studies on A. graveolens seeds were focused
primarily on the bioactivity of crude extracts. Therefore, the primary purpose of the
current research was to isolate and identify the presence of biologically active compounds
not previously examined. In this chapter, we report the cyclooxygenase and
topoisomerase inhibitory and antioxidant activities of compounds 5-10 from A.

graveolens seeds.

63

MATERIALS AND METHODS

General Experimental procedures

lH- and ‘3 C NMR, DEPT, HMQC and HMBCspectra were recorded on Varian INOVA
300 and 500 MHz spectrometers. l3C NMR and DEPT spectra were recorded at 75 and
126 MHz. Chemical shift were recorded in CDCl;;, CD3OD and DMSO-d6, and the values
are reported in 6 (ppm) based on 6 residual of 7.24, 3.3 and 2.29 for 1H NMR and 77, 49
and 39.5 for '3 C NMR for CDCl3, CD30D and DMSO-d6, respectively. Coupling
constants J, are in Hz. EIMS were recorded at 70 eV. The silica gel used for VLC and
MPLC was Merk Silica gel 60 (30-70 pm particle size). For preparative HPLC (LC-20,
Japan Analytical Industry Co., Tokyo) purification, two JAIGEL-ODS, A-343-10 (20
mm x 250 mm, 10 pm, Dychrom, Santa Clara, CA) columns were used in tandem. Peaks
were detected using a UV detector equipped with model D-2500 Chromato-integrator
(Hitachi, Tokyo). The CD analysis of compounds 6 and 7 were performed on JASCO J-
710 CD-ORD spectropolarimeter. Compounds 6 and 7 were dissolved in methanol
separately, and CD was determined at 185-400 nm. All solvents were ACS reagent grade

and were purchased from Aldrich Chemical Co., Inc.

Plant Material

Celery seeds were donated by Asgrow Seed Company, Kalamazoo, Michigan and stored

at —20 0C until extraction.

64

Extraction and Isolation

The ground seeds (905 g) were extracted with hexane followed by MeOH to yield 20 g of
MeOH extract. The MeOH extract (19 g) was stirred with hexane (2 x 400 mL, 15 min)
to yield hexane soluble (7.4 g) and insoluble (11.5 g) fractions. The hexane soluble
fraction (6 g) was further fractionated by VLC followed by MPLC on silica gel using
hexane with increasing amount of acetone and finally with MeOH as eluting solvents.
The fractions collected were: A (404 mg, hexane-acetone, 4:1, 250 mL); B (142 mg,
hexane-acetone, 2:1, 80 mL); C (483 mg, hexane-acetone, 2:1, 100 mL); D (79 mg,
hexane-acetone, 2:1, 120 mL); E (61 mg, hexane-acetone, 1:1, 100 mL); F (89 mg,
hexane-acetone, 1:1, 200 mL); G (35 mg, hexane-acetone, 1:1, 100 mL); H (14 mg, 100%
acetone, 20 mL); 1 (33 mg, 100% acetone, 130 mL), and J (8 mg, 100% MeOH, 250 mL).
Topoisomerase and cyclooxygenase bioassays on fractions A-J revealed that fraction B
was active. The bioactive fraction B (135 mg) was purified by preparative TLC (hexane-
acetone, 20:1 x 3) to yield three major bands: I (48 mg, Rf 0.18); 11 (65 mg, Rf 0.35) and
III (16 mg, Rf 0.50). Band II was biologically active and was further purified by repeated

preparative TLC (hexane-ether 8:1 x 4 and 2:1 x 1) to yield a pure compound 5 (22 mg).

Hexane insoluble fraction (11 g) of the MeOH extract was separated into CHCI3
soluble (2.1g) and insoluble (8.2 g) fractions by stirring it with CHCl3 (2 x 50 mL, 15
min). The CHCl3 soluble fraction (2.1 g) was further separated into hexane soluble (607

mg) and insoluble (1.5 g) fractions. The hexane insoluble fraction was further separated

into methanol-water (75:25 v/v) soluble and insoluble fractions by removing the

65

precipitate by centrifirgation. The aqueous MeOH portion (684 mg) was purified by
preparative HPLC using methanol-water (75:25 v/v) as the mobile phase at a flow rate of
3 mLmin'l. Among the 8 fractions collected, the active fraction B (R. = 48 min, 75 mg)
was further purified by HPLC using methanol-water (60:40 v/v) as the mobile phase at a
flow rate of 2 mL min". This yielded a mixture of compounds 6 and 7 (R. = 65 min, 12
mg) and a pure compound 8 (R. = min, 8.5 mg). The mixture of compounds 6 and 7 was

separated into pure compounds 6 and 7 by HPLC using HZO-THF (90:10 v/v) as the
mobile phase at a flow rate of 4 mL min". The acetylation of 8 was performed according

to the published procedure (Ramsewak et al., 1999a) to yield compound 11.

The CHCI3 insoluble fraction of MeOH extract of A. graveolens seeds (10.2 g)
was partitioned into MeOH-water (75:25 v/v) soluble (8 g) and insoluble fractions (1.6 g)
by removing the precipitate by centrifugation. The aqueous MeOH portion (7.5 g) was
further fractionated by MPLC on C13 silica column. The fractions collected were: A (5.25
g, MeOHszO, 50:50, 100 mL); B (1.59 g, MeOH:HzO, 50:50, 130 mL); C (126.6 mg,
MeOHszO, 70:30, 160 mL); D (47.1 mg, MeOH:HzO, 90:10, 60 mL); B (139.2 mg,
MeOH:HzO, 90:10, 120 mL); F (75.9 mg, MeOH:HzO, 90:10, 100 mL) and G (161.7 mg,
100% MeOH, 225 mL). The bioactive fraction E was purified by preparative HPLC (LC-
20, Japan Analytical Industry Co., Tokyo) using methanol-water (60:40 v/v) as the
mobile phase at the flow rate of 3 mL min”. This yielded bioactive compound 10 (R. =
54 min, 4.5 g). The MPLC fraction B (1.5 g) was separated into MeOH soluble (1.2 g)
and insoluble (200 mg) fractions by centrifugation. The MeOH soluble fraction (1.2 g)

was further fractionated into four fractions by MPLC on C13 silica column with MeOH-

66

H2O (60:40, 540 mL) at 2 mL min'l flow rate. Bioactive fraction four (125 mg) was
purified by preparative HPLC using MeOH-H2O (30:70 v/v) as the mobile phase at the

flow rate of 3 mL min’I to yield pure compound 9 (R. = 84.8 min, 5.5 mg).

Compound 8. pale yellow oil; 1H NMR (CDCI3, 300 MHZ): 6 3.08 (m, 1H, H-3), 3.86
(dd, 1H, J=6, 2.1 Hz, H-8a), 3.88 (s, 3H, H-9), 4.23 (dd, 1H, J=9, 3 Hz, H-8b), 4.72 (d,
1H, J=4 Hz, H-2), 5.60 (b, 1H, NH, exchanged with D20), 6.80 (m, 1H, H-7), 6.85 (m,
1H, H-5), 6.88 (m, 1H, H-4); 13C NMR (CDCI3, 75 MHZ): 6 54.09 (C-3), 55.92 (C-9),

71.62 (C-8), 85.82 (C-2), 108.54 (C-4), 114.21 (C-S), 118.92 (C-7), 132.82 (C-3a),

145.16 (C-7a), 146.64 (C-6).

Acetate of 8 (11). 1H NMR (CDCI3, 300 MHZ): 6 2.04 (s, 3H, -OAc), 2.28 (s, 3H, -OAc),

3.08 (m, 1H, H-3), 3.84 (s, 3H, H-9), 3.92 (dd, 1H, J=9, 3 Hz, H-8a), , 4.26 (dd, 1H, J=9,
3 Hz, H-8b), 4.78 (d, 1H, J=4 Hz, H-2), 6.81 (m, 1H, 11.7). 6.86 (m, 1H, H-5), 6.88 (m,

1H, H-4).

Compound 9. 1H NMR (CD3OD, 5()() MHZ): 6 7.69 (d, 1H, J=8 Hz, H-4), 7.34 (d, 1H,
J=8 Hz, H-7), 7.18 (s, 1H, H-2), 7.10 (m, 1H, H-6), 7.04 (m, 1H, H-5), 3.85 (dd, 1H,
J=9.5, 3.5 Hz, H-9), 3.52 (dd, 1H, J=15.5, 4.5 Hz, H- 8a), 3.15 (dd, 1H, 1:155, 9.5 Hz,
H-8b); 13C NMR (CD3OD, 126 MHZ): a 174.4 (C-lO), 138.42 (C-7a), 128.51 (C-3a),

125.13 (C-2), 122.76 (C-6), 120.12 (C-S), 119.34 (C-4), 112.43 (C-7), 109.62 (C-3),

56.77 (C-9), 28.45 (C-8).

67

Compound 10. Yellow amorphous solid; 1H NMR (CD3OD, 300 MHZ): 6 7.84 (1H, (1,
1:9 Hz, H-5’), 7.48 (1H, s, H-3), 6.84 (1H, d, J=3 Hz, H-8), 6.80 (1H, dd, J=9, 2.4 Hz, H-
6’), 6.62 (1H, d, J=3 Hz, H-6), 6.53 (1H, d, J=2.4 Hz, H-2’), 5.45 (1H, d, J=3 Hz, H-l’”),
5.15 (1H, d, J=6 Hz, H-l”), 3.5-4.1 (11H, m, H-2”, H-3”, H-4”, H-5”, H-6”, H-2’”, H-
4’”, H-5’”), 3.34 (3H, s, H:7’); l3C NMR (DMSO-d6, 125 MHZ): 6 181.83 (C-4), 165.48
(C-4’), 164.35 (C-2), 162.58 (C-5), 161.08 (C-7), 156.85 (C-9), 148.42 (C-3’), 128.57 (C-
l’), 116.62 (C-2’), 116.04 (C-S’), 110.21 (C-6’), 108.68 (C-l’”), 105.30 (C-IO), 102. 73
(C-3), 99.26 (C-8), 98.19 (C-l”), 94.84 (C-6), 79.23 (C-3’”), 76.99 (C-3”), 76.76 (C-2’”),
76.02 (C-5”), 75.70 (C-2”), 73.94 (C-5’”), 69.90 (C-4”), 64.18 (C-4’”), 60.49 (C-6”),

55.90 (C-7’).

Cyclooxygenase Inhibitory Assay

Pure compounds 5-11 were assessed for their cyclooxygenase inhibitory activity at 37 0C
by monitoring the initial rate of 02 uptake using an O2 electrode (Instech Laboratories,
Inc., 5209 Militia Road, Plymouth Meeting PA 19462-1216) (Laneuville et al., 1994;
Meade et al., 1993). Cyclooxygenase-I (COX-I) activities were measured by using
prostaglandin H synthase isozymes-I (PGHS-I) from ram seminal vesicles.
Cyclooxygenase-II (COX-II) activities were measured by using human PGHS-II enzyme
obtained from cloned insect cell lysate. Each assay mixture contained 600 pL of 0.1 M
Tris buffer (pH 7), lmM phenol, 100 pM arachidonic acid and 17 pg hemoglobin.
Cyclooxygenase activities were initiated by the addition of 10 pL of COX-1 or 20 pL of
COX-II enzymes. Instantaneous inhibition was determined by measuring the

cyclooxygenase activity initiated by adding aliquots of microsomal suspensions of

68

PGHS-I (10 pM 02 min'1 cyclooxygenase activity/aliquot) to assay mixtures containing
10 pM arachidonate and 1-500 pg mL'l concentrations of the test compounds. Ibuprofen
and naproxen were assayed at their 1C5.) values, 2.52 pg mL'1 and 2.06 pg mL'l

concentrations, respectively.

Antioxidant Assay

Antioxidant bioassays were performed on crude extracts and pure compounds by analysis
of liposome oxidation using fluorescence spectroscopy (Arora et al., 1998; Arora et al.,
1997). A mixture containing 5 pM of 1-steroyl-2-linoleoyl-sn-glyceroI-3-phosphocholine
(Avanti Polar lipids, Inc., Alabaster, AL) and 5 pM of the fluorescence probe 3-[p-(6-
phenyI)-l,3,5-hexatrienyl]phenylpropionic acid (molecular Probes, Inc., Eugene, OR)
was dried in a foil-covered round-bottom flask under vacuum using a rotary evaporator at
room temperature. MBSE buffer (500 pL), consisting of 0.15 M NaCl, 0.1 mM EDTA
(Ethylenediaminetetraacetic acid di-sodium salt) and 10 mM MOPS [3-(N-morpholine
propane sulfonic acid), adjusted to pH 7] was used to suspend this lipid film. Buffer
solutions were treated with Chelex 100 chelating resin to remove trace metal ions. The
lipid-buffer suspension was subjected to 10 freeze-thaw cycles using a dry ice-EtOH
bath. After the last thaw, the lipid-buffer suspension was then extruded 29 times through
a Liposo Fast extruder (Avestin, Inc., Ottawa, Canada) containing a polycarbonate
membrane with a pore size of 100 nm to produce large unilamellar liposomes (LUVs). A
20 pL aliquot of this liposome suspension was diluted to a final volume of 2 mL in
Chelex 100 treated HEPES buffer (100 pL, pH 7), 1M NaCl (200 pL), N2-sparged water

and DMSO solution containing the test compound (20 pL), vortexed, and placed in the

69

cuvette holder of the spectrophotometer. Peroxidation then was initiated by the addition
of 20 pL of 0.5'mM FeCl2 stock solution to achieve a final concentration of 5 pM of Fe+2
in the presence of test compounds or crude extract dissolved in DMSO. The control
sample did not contain either-Fe+2 or the test compounds. The positive controls BHA,
BHT, TBHQ, and (it-tocopherol (Vitamin E) were all tested at a final concentration of 1.8,
2.2, 1.66 and 4.31 pg mL'l, respectively. Fluorescence intensities of these liposome
solutions were measured at an excitation wavelength of 384 nm for every 3 min over a
period of 21 min using a Turner model 450 digital fluorometer (Barnstead Thermolyne,
Dubuque, IA). The decrease of relative fluorescence intensity with time indicated the rate
of peroxidation. Relative fluorescence was calculated by dividing the fluorescence value

at a given time point (F .) by that at t = 0 min (F0).

Topoisomerase-I and -II Inhibitory Assay

Compounds 5-7 were evaluated for topoisomerase-l and —II inhibitory activities
according to the reported procedure (Chang et al., 1995). The S. cerevisiae mutant cell
cultures, JN394, JN394L1 and JN394t-2-5 used for the topoisomerase inhibitory assays
were cultured in petri dishes containing YPDA medium (20 mL). Two to three mL of
physiological saline solution was added to fully grown plate of each organism and then
suspensions were diluted to obtain 5 x 106 CPU mL". Bioassays were conducted by
spreading 50 pL of the desired cell suspension to petri dishes of the YPDA media.
DMSO or test compound dissolved in DMSO (20 pL) were spotted carefully on the
bioassay plates at varying concentrations. The plates were allowed to dry in a laminar

flow hood and then incubated at 27 0C for 72 h. The zone of inhibition was measured in

70

mm. Minimum inhibitory concentrations (MICmo) for compounds 5-7 were determined

according to the published procedure (Nair et al., 1989).

RESULTS AND DISCUSSION

Compounds 5-7 were isolated and characterized as described in Chapter 3,

Results and Discussion section.

The structure of compound 8 was deduced by using lH and 13 C NMR, DEPT,
HMQC, HMBC, IR and GCMS spectral techniques. lH NMR spectrum of compound 8
gave a singlet, integrated for three protons at 3.88 ppm, was corresponded to a methoxy
group. A broad peak at 5.6 ppm was determined to be an —NH group, as indicated by the
disappearance of this peak with D20 shake in its 1H NMR spectrum. The 13C NMR
spectrum of 8 revealed ten peaks, which were assigned to three aromatic carbons (6
108.5, 114.2, 118.9), one methoxy carbon (6 55.92), three quaternary carbons (6 132.8,
145.2, 146.6), two CH (6 85.8, 54.09) and one CH2 (6 71.6) as indicated by the DEPT

spectrum of 8.

OAc

OH 2 OAc

I2

6

I2

71

Both HMQC and HMBC spectral data provided confirmation of the structure
proposed for compound 8. The important connectivities in HMBC spectrum of 8 were
those observed for C-3a and C-8 (Fig. 4.1). The signal for C-3a correlated to H-3 and H-2
signals while C-8 displayed cross signals to H-2. Also, the correlation of C-2 to H-8a and
H-8b as well as the correlation of C-3 to H-8a, H-8b and H-2 supported the proposed
structure (Fig. 4.1). Three methoxy protons showed correlation to C-6. The presence of
the OH groups in 8 were suggested by a broad peak at 3372 cm'1 in its IR spectrum and
confirmed by the formation of a di-acetate of 8, compound 11. The MS analysis of 8 gave
a weak M ion peak at m/z 195 (9 %). The fragmentation pattern of 8 under EIMS
condition is shown in Scheme-1. The EIMS fragmentation pattern with ions at m/z 73,
151, 163, 193 and 194 was suggestive of the proposed structure for compound 8
(Scheme-1). The NMR analysis of the acetate of 8, compound 11, showed the presence
of two acetoxy groups. The MS of 11 gave peaks at m/z 279 and 280 confirming the
acetylation of two OH groups in 8. Based on all spectral data of the natural product and
its acetate, compound 8 was deduced to be 3-hydroxymethyl-6-methoxy-2,3-dihydro-lH-
indol-2-ol. Compound 8 is a novel natural product.

Figure 4.1. HMBC connectivities in compound 8

H
H C OH
H l ( \/
Vic/ \C/\/OH
”\o ll .1 ‘ C\
H/ \O/ \T/ \n H
H

72

Scheme 1

 

 

+ .
‘ OH
EC...
"5%.
H300 ‘ fl
m/z 195 (16 %)
8 _ H -
ll
OH

/ OH =

+
”:90 ll ch0
m/z 194 (55 °/o)

 

 

 

 

 

 

 

_ H °
+ .
OH
b
O #-
”360111,. H,co
m/z 193 (49 %)
a
V + .
OH
H3O
O
N
H
m/z 163 (9 %)

73

 

C...

m/z 73 (76 %)
OH
+
OH
11
+
i\...

m/z 151 (25 %)

+
cu,

m/z 121 (4 %)

The CHC13 insoluble fraction of MeOH extract (:f A. graveolens seeds was
partitioned into MeOH-water (75:25, v/v) soluble and insoluble fractions by removing the
precipitate by centrifugation. The aqueous MeOH portion was fractionated by MPLC in
C18 silica column and seven fractions, A-G, were collected. The bioactive fraction E was
further purified by preparative HPLC using MeOH-water, which yielded a bioactive
compound 10. The active MPLC fraction B was partitioned into MeOH soluble and
insoluble fractions by centrifugation. The MeOH soluble fraction was further fractionated
into four fractions by MPLC on C13 silica column with MeOH: water (60:40, v/v, 540
mL). Bioactive fraction IV was further purified by preparative HPLC using water-MeOH

(70:30, v/v) as the mobile phase to yield a pure compound 9.

The structure of compound 9 was deduced by detailed analysis of the |H- and 13’C-
NMR spectra. The 1H NMR spectrum of 9 gave two doublets at 6 7.69 and 7.34 and two
multiplets at 6 7.04 and 7.1 suggested a disubstituted aromatic ring in the molecule. A
singlet at 6 7.18 correlated to a carbon peak at 6 125.13 in HMQC and the chemical shift
of C-7a (6 138.42) suggested the presence of heteroatom in a five-membered ring
attached to an aromatic ring. A peak in the 1H NMR spectrum of 9 at 6 10.82 in DMSO-
d6 which corresponded to a carbon peak at 6 174.4 in its 13C NMR spectrum suggested
the presence of a carboxylic acid group in the molecule. The presence of one CH2 and
one CH group next to each other was also confirmed by their splitting pattern in 1H NMR
spectrum, HMQC and HMBC correlation spectra. From the spectral data, compound 9
was characterized as L-tryptophan. lH- and 13 C- NMR spectra of compound 9 are in
agreement with the reported values of L-tryptophan (Lee and Phillips, 1992; Goodnow et

al., 1991; Morales-Rios et al., 1987).

74

 

Detailed analyses of the lH- and 13C- NMR spectra helped to characterize the
structure of 10. In the 1H NMR spectrum of 10, a doublet at 6 7.84 and a doublet of
doublet at 6 6.80 suggested that the B ring in 10 was substituted at C-3’ and C-4’. The
signal at 6 7.48, a singlet integrated for one proton, was assigned to H-3. Another three
aromatic protons (6 6.84, 6.62 and 6.53), all doublets with coupling constants of 3, 3 and
2.4 Hz, respectively, indicated a meta coupling and thus assigned to H-8, H-6 and H-2’,
respectively. Two doublets at 5.45 and 5.15 ppm were assigned to anomeric protons of
fructose and glucose, respectively. The sugar identities were further confirmed by 13 C
NMR experiments. '3 C NMR spectral data of compound 10 are in agreement with the
published data for apiin (Forgacs et al., 1978), a related structure of 10. The difference
between apiin and compound 10 is the presence of a methoxy group at 03’. This
methoxy group appeared as a singlet at 6 3.34 in 1H NMR spectrum corresponded to a
peak at 6 55.9 in 13C NMR spectrum. However, in the published report of apiin by
Forgacs et al. (1978), the carbon assignments for C-7, C-8, C-9 and C-10 are incorrect.
This is evident from the detailed published 13 C NMR values for other related phenolic
glycosides (Ramsewak et al., 1999a). The compound 10 was identified as 7-[3-(3,4-

dihydroxy-4-hydroxymethyl-tetrahydro-furan-2-yloxy)-4,5-dihydroxy-6-hydroxymethyl-

75

Yofil

tetrahydro-pyran-Z-yloxy]-5-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-chromen-4-one.
Compound 10 was reported earlier by Seshadri and Vydeeswaran (1971) and Farooq et
al., (1953) from celery seeds. This is the first report of 1H- and 13C- NMR spectral data

for 3’-methoxy apiin (10).

 

10

Compounds 5-11 were tested for their cyclooxygenase inhibitory activities using
COX-1 and —II enzymes at 500-50 mg/mL (Fig. 4.2-4.8). All test compounds
demonstrated good COX-1 and COX-II inhibitory activities at 500 mg mL". There was
no significant difference in COX-1 and COX-II inhibition at 250, 100 and 50 mg mL'1 for
compound 5. Compounds 6 and 7 showed similar inhibitory activities on COX-1 and -II
enzymes at their respective concentrations. COX-I inhibition decreased with a decrease in
concentration for both compounds, 6 and 7. Compounds 8 and its acetate (11)

demonstrated 57.1 and 31.6 % of COX-1 inhibition and 54.2 and 31.8 % of COX-II

76

inhibition, respectively, when assayed at 500 mg mL". The positive controls ibuprofen
(2.06 pg mL") and naproxen (2.52 pg mL") showed 54.3 and 47.5 % of COX-1
inhibition whereas 39.8 and 41.3 % of COX-II inhibition, respectively (Fig. 4.2-4.8).

Also, compounds 9 and 10 showed 31 and 29 % COX-l inhibition as well as 32 and 21 %

COX-II inhibition at 250 pg mL", respectively.

Figure. 4.2. In vitro dose response of COX-1 and COX-II inhibitory activities of

compound 5. Both ibuprofen and naproxen were tested at 2.06 and

2.52 pg mL", respectively.

COX-l COX-II

6
5

O.

,1

40. f f
30.1 F
zo.‘

10:

“lo Inhibition

 

 

 

 

 

 

 

— 1‘
O C C E E E E C C
CD ~93 2" o. o. a. o. .9 g g E, g g
2 o o a. a. a. a o a a. a a.
a C o o o o *- 9
O 3 a o to o no 3- o. 8 8 8 8

77

Figure. 4.3. In vitro dose response of COX-1 and COX-II inhibitory activities of

compound 6. Both ibuprofen and naproxen were tested at 2.06 and

2.52 pg mL", respectively.

COX-l COX-ll

°/o Inhibition
(A
O

o
l

50ppm h
1

DMSO l
Ibuprofen
Naproxen

500ppm
250ppm
100ppm
Ibuprofen
Naproxen «
500 ppm
250 ppm
100 ppm
50 ppm

Figure. 4.4. In vitro dose response of COX-1 and COX-II inhibitory activities of

compound 7. Both ibuprofen and naproxen were tested at 2.06 and

2.52 pg mL‘l, respectively.

COX-l COX-ll

“lo Inhibition
(.0
o

50ppm I
1

DMSO j
Ibuprofen 1
Naproxen

500ppm I

250ppm

100ppm
Ibuprofen I
Naproxen ‘

500 ppm 1

250 ppm 1.

100 ppm j

50 ppm

78

Figure. 4.5. In vitro dose response of COX-1 and COX—II inhibitory activities of

compound 8. Both ibuprofen and naproxen were tested at 2.06 and

2.52 pg mL'], respectively. .

COX-I COX-II

60
50
40
30
20

DMSO I

50ppm I

100 ppm
50 ppm 1
25 ppm
10 ppm

50°ppm —
100W" ——1
1

Ibuprofen
250ppm
Ibuprofen 1
Naproxen I
500 ppm 1
250 ppm

% Inhibition
o 8
Naproxen _

Figure. 4.6. In vitro dose response of COX-1 and COX-II inhibitory activities of
compound 9. Both ibuprofen and naproxen were tested at 2.06 and
2.52 pg mL", respectively.
COX-l COX-ll
60 1

50;
4o]

30.]
20.
10;
01 — 4.- .. .. if

% Inhibition

DMSO
Ibuprofen
Naproxen

500ppm
250ppm
Ibuprofen
Naproxen
500 ppm
250 ppm

Figure. 4.7. In vitro dose response of COX-1 and COX-II inhibitory activities of

compound 10. Both ibuprofen and naproxen were tested at 2.06 and

2.52 pg mL", respectively.

COX-I COX-II

0
ID

% Inhibition
0.)
O

 

 

 

 

 

 

 

DMSO ]
250ppm
Ibuprofen
Naproxen
500 ppm '
250 ppm

C C
«‘3 92
9 2
°- :1.
3 n:
2 2

Figure. 4.8. In vitro dose response of COX-1 and COX-II inhibitory activities of

compound 11. Both ibuprofen and naproxen were tested at 2.06 and

2.52 pg mL'l, respectively.

 

 

 

 

 

 

 

 

 

cox-1 cox-11
60.
50.1
540 [I
E
a. 151
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$220?
1
10]
Cl fir IT'-1”
C
8.2932223 45138333
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80

Compounds 5-7 demonstrated topoisomerase-I and —II inhibitory activities when tested
on S. cerevisiae mutant strains. S. cerevisiae mutant strains, JN 394 is hypersensitive to
topoisomerase-I poisons, while strain JN394.-. lacks the top-I gene and therefore shows a
lack of response to topoisomerase-I poisons. The strain JN394t-2-5 carries the top-11 gene,
which is resistant to topoisomerase-II poisons but responds to topoisomerase-I poisons.
The MICIO.) determination of compounds 5-7, indicated that compound 5 completely
inhibited topoisomerase-I and —11 activities at 100 pg mL'l, while 6 and 7 inhibited only

topoisomerase-II enzyme at 200 pg mL".

Compound 9, L-tryptophan, exhibited very good antioxidant activity at 250, 125
and 50 pg mL", respectively (Fig. 4.11, 4.12). Compound 10 also inhibited lipid
peroxidation induced by Fe2+ at 250 and 125 pg mL'l concentrations. The rest of the
compounds isolated from A. graveolens seeds did not show significant antioxidant

activities (Fig. 4.11, 4.12).

Earlier reports showed that the inhibitory effect of flavonoids and flavonoids
glycosides on Fe2+ lipid peroxidation was attributed due to their ability to chelate Fe2+
with the formation of inert complexes that are unable to initiate peroxidation (Afanas’ev
et al., 1989). Also, the Fe2+ complexes of flavonoids are considered to retain their free
radical scavenging activities and therefore, can scavenge the free radical intermediate in
lipid peroxidation (Wang et al., 1999). Therefore, the observed antioxidant activity of

compound 10 suggests that it may function as an Fe2+ chelator as well.

81

 

fi—r

Figure 4.9. Antioxidant activities of some commercial antioxidants. Data represent

the mean of duplicate experiments.

—0— DMSO

+ Fe Blank

+TBHQ (1.66 ppm)

Fluorescence
O
O\

—x—BHT (2.2 ppm)

  

--O--BHA(1.8 ppm)

1

l

l

4 T ,,

0 3 6 9 12 15 18 21 +Vit--E(4-3PPm)

Time (min)

Figure 4.10. Antioxidant activity at 21 min for some commercial antioxidants.

Data presented are the mean of duplicate experiments.

 

A 100 -
e 1
g 80 I
E l
E 60 ‘.
0
‘t l
a 40 .
°' 1
:2
_g 20 I
‘5 i
< 0 —
'\ N
\lo 59 $9
0‘ ~23" £9
427' q "x"
(3 A

82

Figure 4.11. Antioxidant activities of compounds 5-11. Data represent the mean of

 

Fluorescence
_o o
A O)

9
N

duplicate experiments.

 

 

Time (min)

- - + - -DMSO

+Fe Blank

- - D - -Compound 5 (100 ppm)

- - 1b - -Compound 6 & 7 (100 ppm)
—i——Compound 8 (100 ppm)
+Compound 9 (250ppm)
-—--—Compound 9 (125ppm)
—a—Compound 9 (50ppm)
+Compound 9 (25ppm)

- - I - -Compound 9(1ppm)
—-A—Compound 10 (250 ppm)
—e—Compound 10 (125ppm)
- - i - -Compound 10 (50ppm)
+Compound 10 (25 ppm)
+Compound 11 (100 ppm)

Figure 4.12. Antioxidant activity at 21 min for compounds 5-11. Data presented are

Antioxidant Activities (%)

the mean of duplicate experiments.

 

Indole like compounds and indole alkaloids found in the plant kingdom have
many constituents whose pharmacological properties are significant to mankind
(Hutchinson, 1980). Several indole alkaloids were reported as antitumor agents (Wall et
al., 1966; Yates et al., 1975). Tryptophan, a precursor for indole biosynthesis is well
documented for its effect on central nervous system and for its therapeutic use in
depression, mania and aggression (Young et al., 1984; Curzon, 1980). Seltzer et al.
(1984) reported that the dietary supplementation of tryptophan combined with a high
carbohydrate and reduced protein and fat diet resulted in a significant reduction in the
pain intensity experienced by chronic pain patients. The observed biological activities of
compounds 8 and 9 supported these findings. The present study also supports the

medicinal use of celery seeds to alleviate gout and arthritic related pain.

84

Chapter 5

Summary and Conclusion

The celery seed oil is one of the important components used by flavor and fragrance
industries. It is also known for its medicinal uses by the folklore in India and many parts
of the world. For these reasons, the celery seed oil has been widely studied for its
chemical composition. The research prior to our present study pertaining to
phytochemistry and bioactivities of crude extracts and pure compounds from A.
graveolens seeds was presented in Chapter 1. The Literature review summarized in
Chapter 1 is focused primarily on the biological activity of crude extracts of A.
graveolens seeds. It was our hypothesis that A. graveolens seeds contain compounds with
an array of biological activities. Our research therefore, has yielded a series of
compounds with antioxidant, topoisomerase and cyclooxygenase inhibitory and other

agricultural related bioactive compounds from A. graveolens seeds.

Bioassay directed isolation and purification of compounds from A. graveolens
seeds are discussed in Chapters 2-4. The structures of purified celery seed compounds
were determined by various spectral techniques such as lH- and 13C- NMR, DEPT,
HMQC, HMBC, FTIR and MS techniques. A. graveolens seeds were sequentially
extracted with hexane and methanol. The separation and purification of hexane extract of
A. graveolens seeds by various chromatographic techniques yielded three biologically
active compounds, B-selinene (1), 3-n-butyl-4,5-dihydrophthalide (2), 5-allyl-2-
methoxyphenol (3) and a triglyceride, 1,3-di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9,l2-

octadecadienoyl]glycerol (4) (Chapter 2). Compounds 3 and 4 were isolated for the first

85

time from A. graveolens seeds. However, compound 4 was not active in our bioassays.
Bioassay-directed isolation and purification of the methanolic extract of A. graveolens
seeds afforded sedanolide (5), senkyunolide-N (6) and senkyunolide-J (7) (Chapter 3).
Both of these compounds, 6 and 7, were isolated for the first time from A. graveolens
seeds. Their structures were elucidated by 1H and 13 C- NMR spectral data and further
confirmed by their CD spectra. A novel natural product, 3-hydroxymethyl-6—methoxy-
2,3-dihydro-1H-indol-2-ol (8) and two additional compounds, L-tryptophan (9) and 3’-
methoxy apiin (10), were also isolated and characterized from the methanolic extract of
A. graveolens seeds (Chapter 4). Isolation and purification of these compounds were
performed by MPLC and repeated preparative HPLC on C13 columns. The structure of
compound 8 was deduced by using IH- and 13C- NMR, DEPT, HMQC, HMBC, FTIR,

and GCMS spectral techniques.

Mosquitocidal, nematicidal, antimicrobial, antioxidant, cyclooxygenase and
topoisomerase inhibitory activities of these pure compounds were also discussed in
Chapters 2-4. B-selinene (1), 3-n-butyl-4,5-dihydrophthalide (2) and 5-allyl-2-
methoxyphenol (3) isolated from A. graveolens seeds, as oily compounds, demonstrated
100 % mortality on fourth-instar A. aegyptii mosquito larvae at 50, 25 and 200 pg mL",
respectively, in 24 h. Compound 2 inhibited the growth of C. albicans, C. kruseii and
topoisomerase-I and —II enzyme activities at 100 pg mL'l. Also, compound 2 displayed
100 % mortality on nematodes, P. redivivus and C. elegans at 12.5 and 50 pg mL",

respectively (Chapter 2).

86

Biologically active compounds, sedanolide (5), senkyunolide-N (6) and
senkyunolide-J (7) were isolated and identified from methanolic extract of A. graveolens
seeds as discussed in Chapter 3. Compounds 5—7 gave 100 % mortality at 25, 100 and
100 pg mL'l, respectively, on the nematode, P. redivivus. Sedanolide (5) showed 100 %
mortality at 50 pg mL'l on nematode, C. elegans, and fourth-instar mosquito larvae, A.

aegyptii. Also, it inhibited the grthh of C. albicans and C. parapsilasis at 100 pg mL".

3-hydroxymethyl-6-methoxy-2,3-dihydro-1H-indol-Z-ol (8), L-tryptophan (9) and
3’-methoxy apiin (10) were identified from methanolic extract of A. graveolens seeds and
evaluated for biological activities (Chapter 4). Cyclooxygenase and topoisomerase
inhibitory and antioxidant activities for compounds 5-10 were discussed in Chapter 4 as
well. Compounds 5-10 displayed 24, 32, 36, 52, 31 and 29 % of COX-1 inhibitory
activity when assayed at 250 pg mL'I at pH-7, respectively. Also, compounds 5-10
demonstrated 24, 29, 25, 40, 32 and 21 % of COX-II inhibition at pH-7 when assayed at
250 pg mL", respectively. The acetylated product (11) of compound 8 also inhibited the
COX-1 and COX-II enzymes at 31 and 28 %, respectively, when tested at 250 pg mL'l
concentration. The activities of these compounds were compared with ibuprofen and
naproxen at 2.06 and 2.52 pg mL", respectively. A dose response study of 5-11 were
carried out for the COX-1 and COX-II inhibitory activities. Compounds 6-8 showed a
dose response in COX-I inhibitory bioassay whereas compound 8 and its acetate (11)

showed a dose response in COX-II assay.

All compounds isolated from methnol extract of A. graveolens seeds in my

research were evaluated for topoisomerase-I and —II enzyme inhibitory activities.

87

Compound 5 exhibited topoisomerase-I and —II inhibitory activities at 100 pg mL'l
where as compounds 6 and 7 exhibited only topoisomerase-II enzyme inhibitory
activities at 200 pg mL’l when tested on S. cerevisiae mutant cell cultures. Compounds
5—11 were also evaluated for antioxidant activity. Only compounds 9 and 10 exhibited

appreciable antioxidant activity at 125 and 250 pg mL'l concentrations, respectively.

Topoisomerase inhibitory and antioxidant activities of crude extracts and pure
compounds isolated from A. graveolens seeds supported the anecdotal claims such as the
anticarcinogenic and chemopreventative properties of celery seeds. Compounds isolated
from A. graveolens seeds showed comparable cyclooxygenase inhibitory activities with
the commercially available drugs. Our in vitro results support the notion of the use of
celery seeds in folk medicine to control gout related pain and in the reduction of
inflammation. The use of A. graveolens seeds as food supplements or nutraceuticals
would be desirable by the public, considering the current trend towards natural alternative

medicines.

Mosquitocidal, nematicidal and antimicrobial affects of phthalides and a variety
of natural products resulted from my research on A. graveolens seeds are new and
therefore warrants further investigation. The crude extract or purified compounds from A.
graveolens seeds have potential for pharmaceutical applications since they exhibited
anticarcinogenic, antiinflammatory and antioxidant activities. Although, these
compounds have potential as nematode managing agents, further examination is required

for their efficacies under field conditions. Determination of mode of action and

88

appropriate formulations of celery seed extracts or seed powder would be worthwhile

endeavors for additional research.

89

yr.-

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